Conjugates of an IL-2 moiety and a polymer

ABSTRACT

Conjugates of an interleukin-2 (“IL-2”) moiety and one or more nonpeptidic, water-soluble polymers are provided. Typically, the non-peptidic, water-soluble polymer is poly(ethylene glycol) or a derivative thereof. Also provided, among other things, are compositions comprising conjugates, methods of making conjugates, methods of administering compositions to an individual, nucleic acid sequences, expression systems, host cells, and methods for preparing IL-moieties.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/884,901, filed Aug. 27, 2013, which is a 35 U.S.C. § 371 applicationof International Application No. PCT/US2011/060408, filed Nov. 11, 2011,designating the United States, which claims the benefit of priorityunder 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No.61/413,236, filed Nov. 12, 2010, the disclosures of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

Among other things, one or more embodiments of the present inventionrelate generally to conjugates comprising an IL-2 moiety (i.e., a moietyhaving at least some activity similar to human IL-2) and a polymer. Inaddition, the invention relates to (among other things) compositionscomprising conjugates, methods for synthesizing conjugates, and methodsof administering a composition.

BACKGROUND OF THE INVENTION

In healthy humans, the immune system can differentiate between healthycells and cancerous cells. Upon identifying a given cell as cancerous,the immune system typically eliminates it. Thus, when the immune systembreaks down or is overwhelmed, cancers can develop resulting from acompromised immune system's inability to differentiate, and theneliminate, cancer cells. In a patient suffering from cancer,administration of an immunomodulatory protein to the patient may helpreturn (at least in part) that patient's immune system back to normal sothat her immune system's ability to eliminate cancer cells returns. Inthis way, the cancer may be slowed or even eliminated.

One such immunomodulatory protein used in the treatment of patientssuffering from certain cancers is interleukin-2. Interleukin-2 (IL-2) isa naturally occurring cytokine that has activity as both a stimulator ofnatural killer cells (NK cells) and as an inducer of T-cellproliferation. In unglycosylated form, IL-2 has a molecular weight ofabout 15,300 Daltons (although IL-2 is found in vivo in variablyglycosylated forms).

A commercially available unglycosylated human recombinant IL-2 product,aldesleukin (available as the PROLEUKIN® brand of des-alanyl-1,serine-125 human interleukin-2 from Prometheus Laboratories Inc., SanDiego Calif.), has been approved for administration to patientssuffering from metastatic renal cell carcinoma and metastatic melanoma.IL-2 has also been suggested for administration in patients sufferingfrom or infected with hepatitis C virus (HCV), human immunodeficiencyvirus (HIV), acute myeloid leukemia, non-Hodgkin's lymphoma, cutaneousT-cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis,breast cancer and bladder cancer.

Even recommended doses of aldesleukin, however, can cause severe sideeffects, including capillary leak syndrome (CLS) and impaired neutrophilfunction. In view of the potential for these severe side effects, andbecause the recommended treatment cycle involves intravenous infusionover fifteen minutes every eight hours for fourteen doses,administration of aldesleukin occurs within a clinical setting.Moreover, the commercial formulation of aldesleukin includes thepresence of sodium dodecyl sulfate, a substance that appears to berequired to maintain optimal activity through conformational stability.See Arakawa et al. (1994) Int. J. Peptide Protein Res. 43:583-587.

Attempts at addressing the toxicity concerns of IL-2 have been tried. Inone approach, formulation approaches have been attempted. See, forexample, U.S. Pat. No. 6,706,289 and international patent applicationpublication WO 02/00243 and WO 99/60128. In other approaches, certainconjugates of IL-2 have been suggested. See, for example, U.S. Pat. Nos.4,766,106, 5,206,344, 5,089,261 and 4,902,502.

Notwithstanding these approaches, however, there remains a need forconjugates of IL-2. Among other things, one or more embodiments of thepresent invention is therefore directed to such conjugates as well ascompositions comprising the conjugates and related methods as describedherein, which are believed to be new and completely unsuggested by theart.

SUMMARY OF THE INVENTION

Accordingly, in one or more embodiments of the invention, a conjugate isprovided, the conjugate comprising a residue of an IL-2 moietycovalently attached to a water-soluble polymer.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising a residue of an IL-2 moiety covalently attachedto a water-soluble polymer, wherein the residue of the IL-2 moiety iscovalently attached to the water-soluble polymer via a releasablelinkage.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising a residue of an IL-2 moiety covalently attachedto a water-soluble polymer, wherein the IL-2 moiety is a precursor IL-2moiety.

In one or more embodiments of the invention, a conjugate is provided,the conjugate comprising a residue of an IL-2 moiety covalently attachedto a water-soluble polymer, wherein the IL-2 moiety is a non-precursorIL-2 moiety.

In one or more embodiments of the invention, a method for delivering aconjugate is provided, the method comprising the step of subcutaneouslyadministering to a patient a composition comprised of a conjugate of aresidue of an IL-2 and a water-soluble polymer.

In one or more embodiments of the invention, an isolated nucleic acidmolecule is provided, the isolated nucleic acid molecule encoding anIL-2 moiety, wherein said nucleic acid molecule includes a sequencehaving substantial (e.g., at least 80%) sequence identify to thesequence set forth in SEQ ID NO: 5.

In one or more embodiments of the invention, an expression vector isprovided, the expression vector (e.g., an in vitro expression vector)comprising a nucleic acid molecule provided herein.

In one or more embodiments of the invention, a host cell is provided,the host cell (e.g., an in vitro host cell) comprising an expressionvector as provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the DNA sequence and resulting amino acid sequence (SEQID NO:5) of a gene whose synthesis is further described in Example 1.

FIG. 2.1 is a representation of a typical chromatogram following cationexchange chromatography of [mPEG2-C2-fmoc-20K]-[rIL-2] preparedfollowing the procedure set forth in Example 2.

FIG. 2.2 is a representation of a chromatogram following reverse phaseHPLC analysis of [mPEG2-C2-fmoc-20K]-[rIL-2], as further described inExample 2.

FIG. 2.3 is a plot of the release profile of various conjugates preparedin accordance with the procedure set forth in Example 2.

FIG. 3.1 is a representation of a typical chromatogram following cationexchange chromatography of [mPEG2-CAC-fmoc-20K]-[rIL-2] preparedfollowing the procedures set forth in Example 3.

FIG. 3.2 is a representation of a chromatogram following reverse phaseHPLC analysis of [mPEG2-CAC-fmoc-20K]-[rIL-2], as further described inExample 3.

FIG. 3.3 is a representation of the results following MALDI-TOF analysisof the various conjugates prepared in accordance with the procedures setforth in Example 3.

FIG. 4.1 is a representation of a typical chromatogram following cationexchange chromatography of [mPEG2-ru-20K]-[rIL-2] prepared following theprocedures set forth in Example 4.

FIG. 4.2 is a representation of a chromatogram following reverse phaseHPLC analysis of [mPEG2-ru-20K]-[rIL-2], as further described in Example4.

FIG. 5 is a representation of a chromatogram following cation exchangechromatography of [mPEG2-ru-40K]-[rIL-2], as further described inExample 5.

FIG. 6 is a representation of a chromatogram following cation exchangechromatography of [mPEG2-ru-4K]-[rIL-2], as further described in Example6.

FIG. 7 shows a plot of the proliferation of CTLL-2 cells in response toaldesleukin and stable [mPEG2-ru-20K]-[rIL-2], as further described inExample 11. Data points are means of one experiment in triplicatedeterminations. Error bars represent standard error of the mean.

FIG. 8 shows a plot of the proliferation of CTLL-2 cells in response toaldesleukin, released and unreleased [mPEG2-C2-fmoc-20K]-[rIL-2] and[mPEG2-CAC-fmoc-20K]-[rIL-2], as further described in Example 11. Datapoints are means of one experiment in triplicate determinations. Errorbars represent standard error of the mean.

FIG. 9 shows a plot of the concentration-time curves following a singleinjection in mice, as further described in Example 12.

FIG. 10 shows a plot of total lesion area (mm²) for several testcompounds as further described in Example 13.

FIG. 11A and FIG. 11B are plots showing time to tumor progression curvesfor tested articles at various administration schemes, as furtherdescribed in Example 14.

DETAILED DESCRIPTION OF THE INVENTION

Before describing one or more embodiments of the present invention indetail, it is to be understood that this invention is not limited to theparticular polymers, synthetic techniques, IL-2 moieties, and the like,as such may vary.

It must be noted that, as used in this specification and the intendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a polymer” includes a single polymer as well as two ormore of the same or different polymers, reference to “an optionalexcipient” refers to a single optional excipient as well as two or moreof the same or different optional excipients, and the like.

In describing and claiming one or more embodiments of the presentinvention, the following terminology will be used in accordance with thedefinitions described below.

“PEG,” “polyethylene glycol” and “poly(ethylene glycol)” as used herein,are interchangeable and encompass any nonpeptidic water-solublepoly(ethylene oxide). Typically, PEGs for use in accordance with theinvention comprise the following structure “—(OCH₂CH₂)_(n)—” where (n)is 2 to 4000. As used herein, PEG also includes“—CH₂CH₂—O(CH₂CH₂O)_(n)—CH₂CH₂—” and “—(OCH₂CH₂)_(n)O—,” depending uponwhether or not the terminal oxygens have been displaced, e.g., during asynthetic transformation. Throughout the specification and claims, itshould be remembered that the term “PEG” includes structures havingvarious terminal or “end capping” groups and so forth. The term “PEG”also means a polymer that contains a majority, that is to say, greaterthan 50%, of —OCH₂CH₂— repeating subunits. With respect to specificforms, the PEG can take any number of a variety of molecular weights, aswell as structures or geometries such as “branched,” “linear,” “forked,”“multifunctional,” and the like, to be described in greater detailbelow.

The terms “end-capped” and “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group, more preferably aC₁₋₁₀ alkoxy group, and still more preferably a C₁₋₅ alkoxy group. Thus,examples of end-capping moieties include alkoxy (e.g., methoxy, ethoxyand benzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. It must be remembered that the end-capping moiety may include oneor more atoms of the terminal monomer in the polymer [e.g., theend-capping moiety “methoxy” in CH₃O(CH₂CH₂O)_(n)— andCH₃(OCH₂CH₂)_(n)—]. In addition, saturated, unsaturated, substituted andunsubstituted forms of each of the foregoing are envisioned. Moreover,the end-capping group can also be a silane. The end-capping group canalso advantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) to which thepolymer is coupled can be determined by using a suitable detector. Suchlabels include, without limitation, fluorescers, chemiluminescers,moieties used in enzyme labeling, colorimetric (e.g., dyes), metal ions,radioactive moieties, and the like. Suitable detectors includephotometers, films, spectrometers, and the like. The end-capping groupcan also advantageously comprise a phospholipid. When the polymer has anend-capping group comprising a phospholipid, unique properties areimparted to the polymer and the resulting conjugate. Exemplaryphospholipids include, without limitation, those selected from the classof phospholipids called phosphatidylcholines. Specific phospholipidsinclude, without limitation, those selected from the group consisting ofdilauroylphosphatidylcholine, dioleylphosphatidylcholine,dipalmitoylphosphatidylcholine, disteroylphosphatidylcholine,behenoylphosphatidylcholine, arachidoylphosphatidylcholine, andlecithin. The end-capping group may also include a targeting moiety,such that the polymer—as well as anything, e.g., an IL-2 moiety,attached thereto—can preferentially localize in an area of interest.

“Non-naturally occurring” with respect to a polymer as described herein,means a polymer that in its entirety is not found in nature. Anon-naturally occurring polymer may, however, contain one or moremonomers or segments of monomers that are naturally occurring, so longas the overall polymer structure is not found in nature.

The term “water soluble” as in a “water-soluble polymer” polymer is anypolymer that is soluble in water at room temperature. Typically, awater-soluble polymer will transmit at least about 75%, more preferablyat least about 95%, of light transmitted by the same solution afterfiltering. On a weight basis, a water-soluble polymer will preferably beat least about 35% (by weight) soluble in water, more preferably atleast about 50% (by weight) soluble in water, still more preferablyabout 70% (by weight) soluble in water, and still more preferably about85% (by weight) soluble in water. It is most preferred, however, thatthe water-soluble polymer is about 95% (by weight) soluble in water orcompletely soluble in water.

Molecular weight in the context of a water-soluble polymer, such as PEG,can be expressed as either a number average molecular weight or a weightaverage molecular weight. Unless otherwise indicated, all references tomolecular weight herein refer to the weight average molecular weight.Both molecular weight determinations, number average and weight average,can be measured using gel permeation chromatography or other liquidchromatography techniques. Other methods for measuring molecular weightvalues can also be used, such as the use of end-group analysis or themeasurement of colligative properties (e.g., freezing-point depression,boiling-point elevation, or osmotic pressure) to determine numberaverage molecular weight or the use of light scattering techniques,ultracentrifugation or viscometry to determine weight average molecularweight. The polymers of the invention are typically polydisperse (i.e.,number average molecular weight and weight average molecular weight ofthe polymers are not equal), possessing low polydispersity values ofpreferably less than about 1.2, more preferably less than about 1.15,still more preferably less than about 1.10, yet still more preferablyless than about 1.05, and most preferably less than about 1.03.

The terms “active,” “reactive” or “activated” when used in conjunctionwith a particular functional group, refers to a reactive functionalgroup that reacts readily with an electrophile or a nucleophile onanother molecule. This is in contrast to those groups that requirestrong catalysts or highly impractical reaction conditions in order toreact (i.e., a “non-reactive” or “inert” group).

As used herein, the term “functional group” or any synonym thereof ismeant to encompass protected forms thereof as well as unprotected forms.

The terms “spacer moiety,” “linkage” and “linker” are used herein torefer to a bond or an atom or a collection of atoms optionally used tolink interconnecting moieties such as a terminus of a polymer segmentand an IL-2 moiety or an electrophile or nucleophile of an IL-2 moiety.The spacer moiety may be hydrolytically stable or may include aphysiologically hydrolyzable or enzymatically degradable linkage. Unlessthe context clearly dictates otherwise, a spacer moiety optionallyexists between any two elements of a compound (e.g., the providedconjugates comprising a residue of IL-2 moiety and water-soluble polymercan be attached directly or indirectly through a spacer moiety).

“Alkyl” refers to a hydrocarbon chain, typically ranging from about 1 to15 atoms in length. Such hydrocarbon chains are preferably but notnecessarily saturated and may be branched or straight chain, althoughtypically straight chain is preferred. Exemplary alkyl groups includemethyl, ethyl, propyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl,3-methylpentyl, and the like. As used herein, “alkyl” includescycloalkyl as well as cycloalkylene-containing alkyl.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, and t-butyl.

“Cycloalkyl” refers to a saturated or unsaturated cyclic hydrocarbonchain, including bridged, fused, or spiro cyclic compounds, preferablymade up of 3 to about 12 carbon atoms, more preferably 3 to about 8carbon atoms. “Cycloalkylene” refers to a cycloalkyl group that isinserted into an alkyl chain by bonding of the chain at any two carbonsin the cyclic ring system.

“Alkoxy” refers to an —OR group, wherein R is alkyl or substitutedalkyl, preferably C₁₋₆ alkyl (e.g., methoxy, ethoxy, propyloxy, and soforth).

The term “substituted” as in, for example, “substituted alkyl,” refersto a moiety (e.g., an alkyl group) substituted with one or morenoninterfering substituents, such as, but not limited to: alkyl, C₃₋₈cycloalkyl, e.g., cyclopropyl, cyclobutyl, and the like; halo, e.g.,fluoro, chloro, bromo, and iodo; cyano; alkoxy, lower phenyl;substituted phenyl; and the like. “Substituted aryl” is aryl having oneor more noninterfering groups as a substituent. For substitutions on aphenyl ring, the substituents may be in any orientation (i.e., ortho,meta, or para).

“Noninterfering substituents” are those groups that, when present in amolecule, are typically nonreactive with other functional groupscontained within the molecule.

“Aryl” means one or more aromatic rings, each of 5 or 6 core carbonatoms. Aryl includes multiple aryl rings that may be fused, as innaphthyl or unfused, as in biphenyl. Aryl rings may also be fused orunfused with one or more cyclic hydrocarbon, heteroaryl, or heterocyclicrings. As used herein, “aryl” includes heteroaryl.

“Heteroaryl” is an aryl group containing from one to four heteroatoms,preferably sulfur, oxygen, or nitrogen, or a combination thereof.Heteroaryl rings may also be fused with one or more cyclic hydrocarbon,heterocyclic, aryl, or heteroaryl rings.

“Heterocycle” or “heterocyclic” means one or more rings of 5-12 atoms,preferably 5-7 atoms, with or without unsaturation or aromatic characterand having at least one ring atom that is not a carbon. Preferredheteroatoms include sulfur, oxygen, and nitrogen.

“Substituted heteroaryl” is heteroaryl having one or more noninterferinggroups as substituents.

“Substituted heterocycle” is a heterocycle having one or more sidechains formed from noninterfering substituents.

An “organic radical” as used herein shall include akyl, substitutedalkyl, aryl, and substituted aryl.

“Electrophile” and “electrophilic group” refer to an ion or atom orcollection of atoms, which may be ionic, having an electrophilic center,i.e., a center that is electron seeking, capable of reacting with anucleophile.

“Nucleophile” and “nucleophilic group” refers to an ion or atom orcollection of atoms that may be ionic having a nucleophilic center,i.e., a center that is seeking an electrophilic center or with anelectrophile.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa bond that reacts with water (i.e., is hydrolyzed) under physiologicalconditions. The tendency of a bond to hydrolyze in water will depend notonly on the general type of linkage connecting two central atoms butalso on the substituents attached to these central atoms. Appropriatehydrolytically unstable or weak linkages include but are not limited tocarboxylate ester, phosphate ester, anhydrides, acetals, ketals,acyloxyalkyl ether, imines, orthoesters, peptides and oligonucleotides.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “hydrolytically stable” linkage or bond refers to a chemical bond,typically a covalent bond, which is substantially stable in water, thatis to say, does not undergo hydrolysis under physiological conditions toany appreciable extent over an extended period of time. Examples ofhydrolytically stable linkages include, but are not limited to, thefollowing: carbon-carbon bonds (e.g., in aliphatic chains), ethers,amides, urethanes, and the like. Generally, a hydrolytically stablelinkage is one that exhibits a rate of hydrolysis of less than about1-2% per day under physiological conditions. Hydrolysis rates ofrepresentative chemical bonds can be found in most standard chemistrytextbooks.

“Pharmaceutically acceptable excipient or carrier” refers to anexcipient that may optionally be included in the compositions of theinvention and that causes no significant adverse toxicological effectsto the patient. “Pharmacologically effective amount,” “physiologicallyeffective amount,” and “therapeutically effective amount” are usedinterchangeably herein to mean the amount of a polymer-(IL-2) moietyconjugate that is needed to provide a desired level of the conjugate (orcorresponding unconjugated IL-2 moiety) in the bloodstream or in thetarget tissue. The precise amount will depend upon numerous factors,e.g., the particular IL-2 moiety, the components and physicalcharacteristics of the therapeutic composition, intended patientpopulation, individual patient considerations, and the like, and canreadily be determined by one skilled in the art, based upon theinformation provided herein.

“Multi-functional” means a polymer having three or more functionalgroups contained therein, where the functional groups may be the same ordifferent. Multi-functional polymeric reagents of the invention willtypically contain from about 3-100 functional groups, or from 3-50functional groups, or from 3-25 functional groups, or from 3-15functional groups, or from 3 to 10 functional groups, or will contain 3,4, 5, 6, 7, 8, 9 or 10 functional groups within the polymer backbone.

The term “IL-2 moiety,” as used herein, refers to a moiety having humanIL-2 activity. The IL-2 moiety will also have at least one electrophilicgroup or nucleophilic group suitable for reaction with a polymericreagent. In addition, the term “IL-2 moiety” encompasses both the IL-2moiety prior to conjugation as well as the IL-2 moiety residue followingconjugation. As will be explained in further detail below, one ofordinary skill in the art can determine whether any given moiety hasIL-2 activity. Proteins comprising an amino acid sequence correspondingto any one of SEQ ID NOs: 1 through 4 is an IL-2 moiety, as well as anyprotein or polypeptide substantially homologous thereto. As used herein,the term “IL-2 moiety” includes such proteins modified deliberately, asfor example, by site directed mutagenesis or accidentally throughmutations. These terms also include analogs having from 1 to 6additional glycosylation sites, analogs having at least one additionalamino acid at the carboxy terminal end of the protein wherein theadditional amino acid(s) includes at least one glycosylation site, andanalogs having an amino acid sequence which includes at least oneglycosylation site. The term includes both natural and recombinantlyproduced moieties.

The term “substantially homologous” means that a particular subjectsequence, for example, a mutant sequence, varies from a referencesequence by one or more substitutions, deletions, or additions, the neteffect of which does not result in an adverse functional dissimilaritybetween the reference and subject sequences. For purposes of the presentinvention, sequences having greater than 80 percent (more preferablygreater than 85 percent, still more preferably greater than 90 percent,with greater than 95 percent being most preferred) homology, equivalentbiological activity (although not necessarily equivalent strength ofbiological activity), and equivalent expression characteristics areconsidered substantially homologous. For purposes of determininghomology, truncation of the mature sequence should be disregarded.Exemplary IL-2 moieties for use herein include those sequences that aresubstantially homologous SEQ ID NO: 2.

The term “fragment” means any protein or polypeptide having the aminoacid sequence of a portion or fragment of an IL-2 moiety, and which hasthe biological activity of IL-2. Fragments include proteins orpolypeptides produced by proteolytic degradation of an IL-2 moiety aswell as proteins or polypeptides produced by chemical synthesis bymethods routine in the art.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of anactive agent (e.g., conjugate), and includes both humans and animals.

“Optional” or “optionally” means that the subsequently describedcircumstance may or may not occur, so that the description includesinstances where the circumstance occurs and instances where it does not.

“Substantially” means nearly totally or completely, for instance,satisfying one or more of the following: greater than 50%, 51% orgreater, 75% or greater, 80% or greater, 90% or greater, and 95% orgreater of the condition.

As used herein, “sequence identity” is determined by comparing thesequence of the reference DNA sequence to that portion of another DNAsequence so aligned so as to maximize overlap between the two sequenceswhile minimizing sequence gaps, wherein any overhanging sequencesbetween the two sequences are ignored. With respect to any sequenceidentity described herein, it is preferred that at least 80%, morepreferred, 85%, yet more preferred 90%, still yet more preferred 95%sequence identity, with 96%, 97%, 98%, and 99% sequence identities beingmost preferred.

Amino acid residues in peptides are abbreviated as follows:Phenylalanine is Phe or F; Leucine is Leu or L; Isoleucine is Ile or I;Methionine is Met or M; Valine is Val or V; Serine is Ser or S; Prolineis Pro or P; Threonine is Thr or T; Alanine is Ala or A; Tyrosine is Tyror Y; Histidine is His or H; Glutamine is Gln or Q; Asparagine is Asn orN; Lysine is Lys or K; Aspartic Acid is Asp or D; Glutamic Acid is Gluor E; Cysteine is Cys or C; Tryptophan is Trp or W; Arginine is Arg orR; and Glycine is Gly or G.

Turning to one or more embodiments of the invention, a conjugate isprovided, the conjugate comprising a residue of an IL-2 moietycovalently attached (either directly or through a spacer moiety) to awater-soluble polymer. The conjugates of the invention will have one ormore of the following features.

The IL-2 Moiety

As previously stated, the conjugate generically comprises a residue ofan IL-2 moiety covalently attached, either directly or through a spacermoiety, to a water-soluble polymer. As used herein, the term “IL-2moiety” shall refer to the IL-2 moiety prior to conjugation as well asto the IL-2 moiety following attachment to a nonpeptidic, water-solublepolymer. It will be understood, however, that when the original IL-2moiety is attached to a nonpeptidic, water-soluble polymer, the IL-2moiety is slightly altered due to the presence of one or more covalentbonds associated with linkage to the polymer(s). Often, this slightlyaltered form of the IL-2 moiety attached to another molecule is referredto a “residue” of the IL-2 moiety.

The IL-2 moiety can be derived from non-recombinant methods and fromrecombinant methods and the invention is not limited in this regard. Inaddition, the IL-2 moiety can be derived from human sources, animalsources, and plant sources.

The IL-2 moiety can be derived non-recombinantly. For example, it ispossible to isolate IL-2 from biological systems and otherwise obtainIL-2 from cultured media. See, for example, the procedures described inU.S. Pat. No. 4,401,756 and in Pauly et al. (1984) J. Immunol Methods75(1):73-84.

The IL-2 moiety can be derived from recombinant methods. See, forexample, U.S. Pat. No. 5,614,185, the disclosure and the Experimentalprovided herein.

Any IL-2 moiety obtained non-recombinant and recombinant approaches canbe used as an IL-2 moiety in preparing the conjugates described herein.

The IL-2 moiety can be expressed in bacterial [e.g., E. coli, see, forexample, Fischer et al. (1995) Biotechnol. Appl. BioIL-2m.21(3):295-311], mammalian [see, for example, Kronman et al. (1992) Gene121:295-304], yeast [e.g., Pichia pastoris, see, for example, Morel etal. (1997) Biochem. J. 328(1):121-129], and plant [see, for example, Moret al. (2001) Biotechnol. Bioeng. 75(3):259-266] expression systems. Theexpression can occur via exogenous expression (when the host cellnaturally contains the desired genetic coding) or via endogenousexpression.

Although recombinant-based methods for preparing proteins can differ,recombinant methods typically involve constructing the nucleic acidencoding the desired polypeptide or fragment, cloning the nucleic acidinto an expression vector, transforming a host cell (e.g., plant,bacteria, yeast, transgenic animal cell, or mammalian cell such asChinese hamster ovary cell or baby hamster kidney cell), and expressingthe nucleic acid to produce the desired polypeptide or fragment. Methodsfor producing and expressing recombinant polypeptides in vitro and inprokaryotic and eukaryotic host cells are known to those of ordinaryskill in the art.

To facilitate identification and purification of the recombinantpolypeptide, nucleic acid sequences that encode for an epitope tag orother affinity binding sequence can be inserted or added in-frame withthe coding sequence, thereby producing a fusion protein comprised of thedesired polypeptide and a polypeptide suited for binding. Fusionproteins can be identified and purified by first running a mixturecontaining the fusion protein through an affinity column bearing bindingmoieties (e.g., antibodies) directed against the epitope tag or otherbinding sequence in the fusion proteins, thereby binding the fusionprotein within the column. Thereafter, the fusion protein can berecovered by washing the column with the appropriate solution (e.g.,acid) to release the bound fusion protein. The recombinant polypeptidecan also be purified by lysing the host cells, separating thepolypeptide, e.g., by ion-exchange chromatography, affinity bindingapproaches, hydrophobic interaction approaches, and thereafter identifyby MALDI or western blot, and collecting the polypeptide. These andother methods for identifying and purifying recombinant polypeptides areknown to those of ordinary skill in the art. In one or more embodimentsof the invention, however, the IL-2 moiety is not in the form of afusion protein.

Depending on the system used to express proteins having IL-2 activity,the IL-2 moiety can be unglycosylated or glycosylated and either may beused. That is, the IL-2 moiety can be unglycosylated or the IL-2 moietycan be glycosylated. In one or more embodiments of the invention, theIL-2 moiety is unglycosylated.

The IL-2 moiety can advantageously be modified to include and/orsubstitute one or more amino acid residues such as, for example, lysine,cysteine and/or arginine, in order to provide facile attachment of thepolymer to an atom within the side chain of the amino acid. An exampleof substitution of an IL-2 moiety is described in U.S. Pat. No.5,206,344. In addition, the IL-2 moiety can be modified to include anon-naturally occurring amino acid residue. Techniques for adding aminoacid residues and non-naturally occurring amino acid residues are wellknown to those of ordinary skill in the art. Reference is made to J.March, Advanced Organic IL-2mistry: Reactions Mechanisms and Structure,4th Ed. (New York: Wiley-Interscience, 1992).

In addition, the IL-2 moiety can advantageously be modified to includeattachment of a functional group (other than through addition of afunctional group-containing amino acid residue). For example, the IL-2moiety can be modified to include a thiol group. In addition, the IL-2moiety can be modified to include an N-terminal alpha carbon. Inaddition, the IL-2 moiety can be modified to include one or morecarbohydrate moieties. In addition, the IL-2 moiety can be modified toinclude an aldehyde group. In addition, the IL-2 moiety can be modifiedto include a ketone group. In some embodiments of the invention, it ispreferred that the IL-2 moiety is not modified to include one or more ofa thiol group, an N-terminal alpha carbon, carbohydrate, adehyde groupand ketone group.

Exemplary IL-2 moieties are described in the literature and in, forexample, U.S. Pat. Nos. 5,116,943, 5,153,310, 5,635,597, 7,101,965 and7,567,215 and U.S. Patent Application Publication Nos. 2010/0036097 and2004/0175337. Preferred IL-2 moieties include those having an amino acidsequence comprising sequences selected from the group consisting of SEQID NOs: 1 through 4, and sequences substantially homologous thereto. Apreferred IL-2 moiety has the amino acid sequence corresponding to SEQID NO: 3.

In some instances, the IL-2 moiety will be in a “monomer” form, whereina single expression of the corresponding peptide is organized into adiscrete unit. In other instances, the IL-2 moiety will be in the formof a “dimer” (e.g., a dimer of recombinant IL-2) wherein two monomerforms of the protein are associated (e.g., by disulfide bonding) to eachother. For example, in the context of a dimer of recombinant human IL-2,the dimer may be in the form of two monomers associated to each other bya disulfide bond formed from each monomer's Cys125 residue.

In addition, precursor forms IL-2 can be used as the IL-2 moiety. Anexemplary precursor form of IL-2 has the sequence of SEQ ID NO: 1.

Truncated versions, hybrid variants, and peptide mimetics of any of theforegoing sequences can also serve as the IL-2 moiety. Biologicallyactive fragments, deletion variants, substitution variants or additionvariants of any of the foregoing that maintain at least some degree ofIL-2 activity can also serve as an IL-2 moiety.

For any given peptide or protein moiety, it is possible to determinewhether that moiety has IL-2 activity. Various methods for determiningthe in vitro IL-2 activity are described in the art. An exemplaryapproach is the CTTL-2 cell proliferation assay described in theexperimental below. An exemplary approach is described in Moreau et al.(1995) Mol. Immunol. 32:1047-1056). Briefly, in a non-specific bindingassay, a proposed IL-2 moiety is allowed to preincubate for one hour at4° C. in the presence of a cell line bearing a receptor of IL-2.Thereafter, ¹²⁵I-labelled IL-2 is allowed to incubate in the system forthree hours at 4° C. Data is expressed as % inhibitory capacity of theproposed IL-2 moiety activity versus wild-type IL-2. Other methodologiesknown in the art can also be used to assess IL-2 function, includingelectrometry, spectrophotometry, chromatography, and radiometricmethodologies.

The Water-Soluble Polymer

As previously discussed, each conjugate comprises an IL-2 moietyattached to a water-soluble polymer. With respect to the water-solublepolymer, the water-soluble polymer is nonpeptidic, nontoxic,non-naturally occurring and biocompatible. With respect tobiocompatibility, a substance is considered biocompatible if thebeneficial effects associated with use of the substance alone or withanother substance (e.g., an active agent such as an IL-2 moiety) inconnection with living tissues (e.g., administration to a patient)outweighs any deleterious effects as evaluated by a clinician, e.g., aphysician. With respect to non-immunogenicity, a substance is considerednon-immunogenic if the intended use of the substance in vivo does notproduce an undesired immune response (e.g., the formation of antibodies)or, if an immune response is produced, that such a response is notdeemed clinically significant or important as evaluated by a clinician.It is particularly preferred that the nonpeptidic water-soluble polymeris biocompatible and non-immunogenic.

Further, the polymer is typically characterized as having from 2 toabout 300 termini. Examples of such polymers include, but are notlimited to, poly(alkylene glycols) such as polyethylene glycol (“PEG”),poly(propylene glycol) (“PPG”), copolymers of ethylene glycol andpropylene glycol and the like, poly(oxyethylated polyol), poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(α-hydroxy acid),poly(vinyl alcohol), polyphosphazene, polyoxazolines (“POZ”) (which aredescribed in WO 2008/106186), poly(N-acryloylmorpholine), andcombinations of any of the foregoing.

The water-soluble polymer is not limited to a particular structure andcan be linear (e.g., an end capped, e.g., alkoxy PEG or a bifunctionalPEG), branched or multi-armed (e.g., forked PEG or PEG attached to apolyol core), a dendritic (or star) architecture, each with or withoutone or more degradable linkages. Moreover, the internal structure of thewater-soluble polymer can be organized in any number of different repeatpatterns and can be selected from the group consisting of homopolymer,alternating copolymer, random copolymer, block copolymer, alternatingtripolymer, random tripolymer, and block tripolymer.

Typically, activated PEG and other activated water-soluble polymers(i.e., polymeric reagents) are activated with a suitable activatinggroup appropriate for coupling to a desired site on the IL-2 moiety.Thus, a polymeric reagent will possess a reactive group for reactionwith the IL-2 moiety. Representative polymeric reagents and methods forconjugating these polymers to an active moiety are known in the art andfurther described in Zalipsky, S., et al., “Use of FunctionalizedPoly(Ethylene Glycols) for Modification of Polypeptides” in PolyethyleneGlycol Chemistry: Biotechnical and Biomedical Applications, J. M.Harris, Plenus Press, New York (1992), and in Zalipsky (1995) AdvancedDrug Reviews 16:157-182. Exemplary activating groups suitable forcoupling to an IL-2 moiety include hydroxyl, maleimide, ester, acetal,ketal, amine, carboxyl, aldehyde, aldehyde hydrate, ketone, vinylketone, thione, thiol, vinyl sulfone, hydrazine, among others.

Preferably, the polymeric reagent used to prepare the conjugatesdescribed herein is prepared without the use of phosgene. Such anapproach stands in contrast to, for example, the disclosure set forth inU.S. Pat. No. 4,902,502, which specifically describes forming achloroformate and subsequent used to form a PEG active ester, which isthen reacted with IL-2. Use of phosgene leads to the formation ofhydrogen chloride, which can lead to chain cleavage in the polymer,thereby increasing impurities, which may not be able to be removed usingconventional techniques. Thus, without wishing to be bound by theory,IL-2 moiety conjugates prepared from polymeric reagents formed withoutthe use of phosgene provides higher quality compositions that aresubstantially absent polymer chain degradation products. Also, in one ormore embodiments, the spacer moiety between the water-soluble polymerand the IL-2 moiety is not a carbamate-containing spacer moiety.

Typically, the weight-average molecular weight of the water-solublepolymer in the conjugate is from about 100 Daltons to about 150,000Daltons. Exemplary ranges, however, include weight-average molecularweights in the range of greater than 5,000 Daltons to about 100,000Daltons, in the range of from about 6,000 Daltons to about 90,000Daltons, in the range of from about 10,000 Daltons to about 85,000Daltons, in the range of greater than 10,000 Daltons to about 85,000Daltons, in the range of from about 20,000 Daltons to about 85,000Daltons, in the range of from about 53,000 Daltons to about 85,000Daltons, in the range of from about 25,000 Daltons to about 120,000Daltons, in the range of from about 29,000 Daltons to about 120,000Daltons, in the range of from about 35,000 Daltons to about 120,000Daltons, and in the range of from about 40,000 Daltons to about 120,000Daltons. For any given water-soluble polymer, PEGs having a molecularweight in one or more of these ranges are preferred.

Exemplary weight-average molecular weights for the water-soluble polymerinclude about 100 Daltons, about 200 Daltons, about 300 Daltons, about400 Daltons, about 500 Daltons, about 600 Daltons, about 700 Daltons,about 750 Daltons, about 800 Daltons, about 900 Daltons, about 1,000Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200 Daltons,about 2,500 Daltons, about 3,000 Daltons, about 4,000 Daltons, about4,400 Daltons, about 4,500 Daltons, about 5,000 Daltons, about 5,500Daltons, about 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons,about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons, about 14,000Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000Daltons, about 70,000 Daltons, and about 75,000 Daltons. Branchedversions of the water-soluble polymer (e.g., a branched 40,000 Daltonwater-soluble polymer comprised of two 20,000 Dalton polymers) having atotal molecular weight of any of the foregoing can also be used. In oneor more embodiments, the conjugate will not have any PEG moietiesattached, either directly or indirectly, with a PEG having a weightaverage molecular weight of less than about 6,000 Daltons.

When used as the polymer, PEGs will typically comprise a number of(OCH₂CH₂) monomers [or (CH₂CH₂O) monomers, depending on how the PEG isdefined]. As used throughout the description, the number of repeatingunits is identified by the subscript “n” in “(OCH₂CH₂)_(n).” Thus, thevalue of (n) typically falls within one or more of the following ranges:from 2 to about 3400, from about 100 to about 2300, from about 100 toabout 2270, from about 136 to about 2050, from about 225 to about 1930,from about 450 to about 1930, from about 1200 to about 1930, from about568 to about 2727, from about 660 to about 2730, from about 795 to about2730, from about 795 to about 2730, from about 909 to about 2730, andfrom about 1,200 to about 1,900. For any given polymer in which themolecular weight is known, it is possible to determine the number ofrepeating units (i.e., “n”) by dividing the total weight-averagemolecular weight of the polymer by the molecular weight of the repeatingmonomer.

One particularly preferred polymer for use in the invention is anend-capped polymer, that is, a polymer having at least one terminuscapped with a relatively inert group, such as a lower C₁₋₆ alkoxy group,although a hydroxyl group can also be used. When the polymer is PEG, forexample, it is preferred to use a methoxy-PEG (commonly referred to asmPEG), which is a linear form of PEG wherein one terminus of the polymeris a methoxy (—OCH₃) group, while the other terminus is a hydroxyl orother functional group that can be optionally chemically modified.

In one form useful in one or more embodiments of the present invention,free or unbound PEG is a linear polymer terminated at each end withhydroxyl groups:HO—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OH,wherein (n) typically ranges from zero to about 4,000.

The above polymer, alpha-, omega-dihydroxylpoly(ethylene glycol), can berepresented in brief form as HO-PEG-OH where it is understood that the-PEG- symbol can represent the following structural unit:—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—,wherein (n) is as defined as above.

Another type of PEG useful in one or more embodiments of the presentinvention is methoxy-PEG-OH, or mPEG in brief, in which one terminus isthe relatively inert methoxy group, while the other terminus is ahydroxyl group. The structure of mPEG is given below.CH₃O—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—OHwherein (n) is as described above.

Multi-armed or branched PEG molecules, such as those described in U.S.Pat. No. 5,932,462, can also be used as the PEG polymer. For example,PEG can have the structure:

wherein:

poly_(a) and poly_(b) are PEG backbones (either the same or different),such as methoxy poly(ethylene glycol);

R″ is a nonreactive moiety, such as H, methyl or a PEG backbone; and

P and Q are nonreactive linkages. In a preferred embodiment, thebranched PEG polymer is methoxy poly(ethylene glycol) disubstitutedlysine. Depending on the specific IL-2 moiety used, the reactive esterfunctional group of the disubstituted lysine may be further modified toform a functional group suitable for reaction with the target groupwithin the IL-2 moiety.

In addition, the PEG can comprise a forked PEG. An example of a forkedPEG is represented by the following structure:

wherein: X is a spacer moiety of one or more atoms and each Z is anactivated terminal group linked to CH by a chain of atoms of definedlength. International Patent Application Publication WO 99/45964discloses various forked PEG structures capable of use in one or moreembodiments of the present invention. The chain of atoms linking the Zfunctional groups to the branching carbon atom serve as a tetheringgroup and may comprise, for example, alkyl chains, ether chains, esterchains, amide chains and combinations thereof.

The PEG polymer may comprise a pendant PEG molecule having reactivegroups, such as carboxyl, covalently attached along the length of thePEG rather than at the end of the PEG chain. The pendant reactive groupscan be attached to the PEG directly or through a spacer moiety, such asan alkylene group.

In addition to the above-described forms of PEG, the polymer can also beprepared with one or more weak or degradable linkages in the polymer,including any of the above-described polymers. For example, PEG can beprepared with ester linkages in the polymer that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:-PEG-CO₂-PEG-+H₂O→-PEG-CO₂H+HO-PEG-

Other hydrolytically degradable linkages, useful as a degradable linkagewithin a polymer backbone and/or as a degradable linkage to an IL-2moiety, include: carbonate linkages; imine linkages resulting, forexample, from reaction of an amine and an aldehyde (see, e.g., Ouchi etal. (1997) Polymer Preprints 38(1):582-3); phosphate ester linkagesformed, for example, by reacting an alcohol with a phosphate group;hydrazone linkages which are typically formed by reaction of a hydrazideand an aldehyde; acetal linkages that are typically formed by reactionbetween an aldehyde and an alcohol; orthoester linkages that are, forexample, formed by reaction between a formate and an alcohol; amidelinkages formed by an amine group, e.g., at an end of a polymer such asPEG, and a carboxyl group of another PEG chain; urethane linkages formedfrom reaction of, e.g., a PEG with a terminal isocyanate group and a PEGalcohol; peptide linkages formed by an amine group, e.g., at an end of apolymer such as PEG, and a carboxyl group of a peptide; andoligonucleotide linkages formed by, for example, a phosphoramiditegroup, e.g., at the end of a polymer, and a 5′ hydroxyl group of anoligonucleotide.

Such optional features of the conjugate, i.e., the introduction of oneor more degradable linkages into the polymer chain or to the IL-2moiety, may provide for additional control over the final desiredpharmacological properties of the conjugate upon administration. Forexample, a large and relatively inert conjugate (i.e., having one ormore high molecular weight PEG chains attached thereto, for example, oneor more PEG chains having a molecular weight greater than about 10,000,wherein the conjugate possesses essentially no bioactivity) may beadministered, which is released to generate a bioactive conjugatepossessing a portion of the original PEG chain. In this way, theproperties of the conjugate can be more effectively tailored to balancethe bioactivity of the conjugate over time.

The water-soluble polymer associated with the conjugate can also be“releasable.” That is, the water-soluble polymer releases (eitherthrough hydrolysis, enzymatic processes, catalytic processes orotherwise), thereby resulting in the unconjugated IL-2 moiety. In someinstances, releasable polymers detach from the IL-2 moiety in vivowithout leaving any fragment of the water-soluble polymer. In otherinstances, releasable polymers detach from the IL-2 moiety in vivoleaving a relatively small fragment (e.g., a succinate tag) from thewater-soluble polymer. An exemplary cleavable polymer includes one thatattaches to the IL-2 moiety via a carbonate linkage.

Those of ordinary skill in the art will recognize that the foregoingdiscussion concerning nonpeptidic and water-soluble polymer is by nomeans exhaustive and is merely illustrative, and that all polymericmaterials having the qualities described above are contemplated. As usedherein, the term “polymeric reagent” generally refers to an entiremolecule, which can comprise a water-soluble polymer segment and afunctional group.

As described above, a conjugate of the invention comprises awater-soluble polymer covalently attached to an IL-2 moiety. Typically,for any given conjugate, there will be one to three water-solublepolymers covalently attached to one or more moieties having IL-2activity. In some instances, however, the conjugate may have 1, 2, 3, 4,5, 6, 7, 8 or more water-soluble polymers individually attached to anIL-2 moiety. Any given water-soluble polymer may be covalently attachedto either an amino acid of the IL-2 moiety, or, when the IL-2 moiety is(for example) a glycoprotein, to a carbohydrate of the IL-2 moiety.Attachment to a carbohydrate may be carried out, e.g., using metabolicfunctionalization employing sialic acid-azide chemistry [Luchansky etal. (2004) Biochemistry 43(38):12358-12366] or other suitable approachessuch as the use of glycidol to facilitate the introduction of aldehydegroups [Heldt et al. (2007) European Journal of Organic Chemistry32:5429-5433].

The particular linkage within the moiety having IL-2 activity and thepolymer depends on a number of factors. Such factors include, forexample, the particular linkage chemistry employed, the particular IL-2moiety, the available functional groups within the IL-2 moiety (eitherfor attachment to a polymer or conversion to a suitable attachmentsite), the presence of additional reactive functional groups within theIL-2 moiety, and the like.

The conjugates of the invention can be, although not necessarily,prodrugs, meaning that the linkage between the polymer and the IL-2moiety is releasable to allow release of the parent moiety. Exemplaryreleasable linkages include carboxylate ester, phosphate ester, thiolester, anhydrides, acetals, ketals, acyloxyalkyl ether, imines,orthoesters, peptides and oligonucleotides. Such linkages can be readilyprepared by appropriate modification of either the IL-2 moiety (e.g.,the carboxyl group C terminus of the protein, or a side chain hydroxylgroup of an amino acid such as serine or threonine contained within theprotein, or a similar functionality within the carbohydrate) and/or thepolymeric reagent using coupling methods commonly employed in the art.Most preferred, however, are releaseable linkages that are readilyformed by reaction of a suitably activated polymer with a non-modifiedfunctional group contained within the moiety having IL-2 activity.

Alternatively, a hydrolytically stable linkage, such as an amide,urethane (also known as carbamate), amine, thioether (also known assulfide), or urea (also known as carbamide) linkage can also be employedas the linkage for coupling the IL-2 moiety. Again, a preferredhydrolytically stable linkage is an amide. In one approach, awater-soluble polymer bearing an activated ester can be reacted with anamine group on the IL-2 moiety to thereby result in an amide linkage.

The conjugates (as opposed to an unconjugated IL-2 moiety) may or maynot possess a measurable degree of IL-2 activity. That is to say, apolymer-IL-2 moiety conjugate in accordance with the invention willpossesses anywhere from about 0.1% to about 100% of the bioactivity ofthe unmodified parent IL-2 moiety. In some instances, the polymer-IL-2moiety conjugates may have greater than 100% bioactivity of theunmodified parent IL-2 moiety. Preferably, conjugates possessing littleor no IL-2 activity contain a hydrolyzable linkage connecting thepolymer to the moiety, so that regardless of the lack (or relativelylack) of activity in the conjugate, the active parent molecule (or aderivative thereof) is released upon aqueous-induced cleavage of thehydrolyzable linkage. Such activity may be determined using a suitablein-vivo or in-vitro model, depending upon the known activity of theparticular moiety having IL-2 activity employed.

For conjugates possessing a hydrolytically stable linkage that couplesthe moiety having IL-2 activity to the polymer, the conjugate willtypically possess a measurable degree of bioactivity. For instance, suchconjugates are typically characterized as having a bioactivitysatisfying one or more of the following percentages relative to that ofthe unconjugated IL-2 moiety: at least about 2%, at least about 5%, atleast about 10%, at least about 15%, at least about 25%, at least about30%, at least about 40%, at least about 50%, at least about 60%, atleast about 80%, at least about 85%, at least about 90%, at least about95%, at least about 97%, at least about 100%, and more than 105% (whenmeasured in a suitable model, such as those well known in the art).Preferably, conjugates having a hydrolytically stable linkage (e.g., anamide linkage) will possess at least some degree of the bioactivity ofthe unmodified parent moiety having IL-2 activity.

Exemplary conjugates in accordance with the invention will now bedescribed. Typically, such an IL-2 moiety is expected to share (at leastin part) a similar amino acid sequence as the sequence provided in atleast one of SEQ ID NOs: 1 through 4. Thus, while reference will be madeto specific locations or atoms within SEQ ID NOs: 1 through 4, such areference is for convenience only and one having ordinary skill in theart will be able to readily determine the corresponding location or atomin other moieties having IL-2 activity. In particular, the descriptionprovided herein for native human IL-2 is often applicable to fragments,deletion variants, substitution variants or addition variants of any ofthe foregoing.

Amino groups on IL-2 moieties provide a point of attachment between theIL-2 moiety and the water-soluble polymer. Using the amino acid sequenceprovided in SEQ ID NOs: 1 through 4, it is evident that there areseveral lysine residues in each having an ε-amino acid that may beavailable for conjugation. Further, the N-terminal amine of any proteincan also serve as a point of attachment.

There are a number of examples of suitable polymeric reagents useful forforming covalent linkages with available amines of an IL-2 moiety.Specific examples, along with the corresponding conjugate, are providedin Table 1, below. In the table, the variable (n) represents the numberof repeating monomeric units and “—NH-(IL-2)” represents the residue ofthe IL-2 moiety following conjugation to the polymeric reagent. Whileeach polymeric portion [e.g., (OCH₂CH₂)_(n) or (CH₂CH₂O)_(n)] presentedin Table 1 terminates in a “CH₃” group, other groups (such as H andbenzyl) can be substituted therefor.

TABLE 1 Amine-Selective Polymeric Reagents and the IL-2 Moiety ConjugateFormed Therefrom Polymeric Reagent Corresponding Conjugate

Carbamate Linkage mPEG-Oxycarbonylimidazole Reagents

Carbamate Linkage mPEG Nitrophenyl Reagents

Carbamate Linkage mPEG-Trichlorophenyl Carbonate Reagents

Amide Linkage mPEG-Succinimidyl Reagents

Amide Linkages Homobifunctional PEG-Succinimidyl Reagents

Amide Linkage Heterobifunctional PEG-Succinimidyl Reagents

Amide Linkage mPEG-Succinimidyl Reagents

Amide Linkage mPEG-Succinimdyl Reagents

Amide Linkage mPEG Succinimidyl Reagents

Amide Linkage mPEG-Succinimidyl Reagents

Carbamate Linkage mPEG-Benzotriazole Carbonate Reagents

Carbamate Linkage mPEg-Succinimidyl Reagents

Amide Linkage mPEG-Succinimidyl Reagents

Amide Linkage mPEG Succinimidyl Reagents

Amide Linkage Branched mPEG2-N-Hydroxysuccinimide Reagents

Branched mPEG2-Aldehyde Reagents Secondary Amine Linkage

Amide Linkage mPEG-Succinimidyl Reagents

Amide Linkage mPEG-Succinimidyl Reagents

Amide Linkages Homobifunctional PEG-Succinimidyl Reagents

Amide Linkage mPEG-Succinimidyl Reagents

Amide Linkages Homobifunctional PEG-Succinimidyl Propionate Reagents

Amide Linkage mPEG-Succinimidyl Reagents

Amide Linkage Branched mPEG2-N-Hydroxysuccinimide Reagents

Amide Linkage Branched mPEG2-N-Hydroxysuccinimide Reagents

mPEG-Thioester Reagents Amide Linkage (typically to IL-2 moiety havingan N-terminal cysteine or histidine)

Homobifunctional PEG Propionaldehyde Reagents Secondary Amine Linkages

H₃C—(OCH₂CH₂)_(n)—CH₂CH₂—CH₂—NH—(IL-2) Secondary Amine Linkage mPEGPropionaldehyde Reagents

Homobifunctional PEG Butyraldehyde Reagents Secondary Amine Linkages

H₃C—(OCH₂CH₂)_(n)—O—CH₂CH₂CH₂—CH₂—NH—(IL-2) Secondary Amine Linkage mPEGButryaldehyde Reagents

mPEG Butryaldehyde Reagents

Secondary Amine Linkage

Hobobifunctional PEG Butryaldehyde Reagents Secondary Amine Linkages

Branched mPEG2 Butyraldehyde Reagents Secondary Amine Linkage

Branched mPEG2 Butyraldehyde Reavents Secondary Amine Linkage

Secondary Amine Linkage mPEG Acetal Reagents

mPEG Piperidone Reagents Secondary Amine Linkage (to a secondary carbon)

mPEG Methylketone Reagents secondary amine linkage (to a secondarycarbon)

H₃CO—(CH₂CH₂O)_(n)—CH₂CH₂—NH—(IL-2) Secondary Amine Linkage mPEGTresylate Reagents

mPEG Maleimide Reagents Secondary Amine Linkage (under certain reactionconditions such as pH > 8)

mPEG Maleimide Reagents Secondary Amine Linkage (under certain reactionconditions such as pH > 8)

Secondary Amine Linkage mPEG Maleimide Reagents (under certain reactionconditions such as pH > 8)

Secondary Amine Linkages mPEG Forked Maleimide Reagents (under certainreaction conditions such as pH > 8)

Secondary Amine Linkage branched mPEG2 Maleimide Reagents (under certainreaction conditions such as pH > 8)

mPEG Epoxide Reagents Secondary Amine Linkage (under certain reactionconditions such as pH > 8)

Branched mPEG Derivative Releasable Linkage

Branched mPEG Derivative Releaseable Linkage

Branched mPEG Derivative Releaseable Linkage

Branched mPEG Derivative Releaseable Linkage

Branched mPEG Derivative Releaseable Linkage

Releaseable Linkage Branched mPEG Derivative

Branched mPEG Derivative Releaseable Linkage

Conjugation of a polymeric reagent to an amino group of an IL-2 moietycan be accomplished by a variety of techniques. In one approach, an IL-2moiety can be conjugated to a polymeric reagent functionalized with asuccinimidyl derivative (or other activated ester group, whereinapproaches similar to those described for these alternative activatedester group-containing polymeric reagents can be used). In thisapproach, the polymer bearing a succinimidyl derivative can be attachedto the IL-2 moiety in an aqueous media at a pH of 7 to 9.0, althoughusing different reaction conditions (e.g., a lower pH such as 6 to 7, ordifferent temperatures and/or less than 15° C.) can result in theattachment of the polymer to a different location on the IL-2 moiety. Inaddition, an amide linkage can be formed by reacting an amine-terminatednonpeptidic, water-soluble polymer with an IL-2 moiety bearing anactivating a carboxylic acid group.

Exemplary conjugates are encompassed within the following structure

wherein:

(n) is an integer having a value of from 2 to 4000;

X is a spacer moiety;

R¹ is an organic radical; and

IL-2 is a residue of an IL-2 moiety.

Exemplary conjugates are encompassed by the following structure:

wherein (n) an integer having a value of from 2 to 4000 and IL-2 is aresidue of an IL-2 moiety.

Typical of another approach useful for conjugating the IL-2 moiety to apolymeric reagent is use of reductive amination to conjugate a primaryamine of an IL-2 moiety with a polymeric reagent functionalized with aketone, aldehyde or a hydrated form thereof (e.g., ketone hydrate,aldehyde hydrate). In this approach, the primary amine from the IL-2moiety reacts with the carbonyl group of the aldehyde or ketone (or thecorresponding hydroxyl-containing group of a hydrated aldehyde orketone), thereby forming a Schiff base. The Schiff base, in turn, canthen be reductively converted to a stable conjugate through use of areducing agent such as sodium borohydride. Selective reactions (e.g., atthe N-terminus) are possible, particularly with a polymer functionalizedwith a ketone or an alpha-methyl branched aldehyde and/or under specificreaction conditions (e.g., reduced pH).

Exemplary conjugates of the invention wherein the water-soluble polymeris in a branched form include those wherein the water-soluble polymer isencompassed within the following structure:

wherein each (n) is independently an integer having a value of from 2 to4000.

Exemplary conjugates of the invention are encompassed within thefollowing structure:

wherein:

each (n) is independently an integer having a value of from 2 to 4000;

X is spacer moiety;

(b) is an integer having a value 2 through 6;

(c) is an integer having a value 2 through 6;

R², in each occurrence, is independently H or lower alkyl; and

IL-2 is a residue of an IL-2 moiety.

Exemplary conjugates of the invention are encompassed within thefollowing structure:

wherein:

each (n) is independently an integer having a value of from 2 to 4000;and

IL-2 is a residue of an IL-2 moiety.

Other exemplary conjugates of the invention are encompassed withinfollowing structure:

wherein:

each (n) is independently an integer having a value of from 2 to 4000;

(a) is either zero or one;

X, when present, is a spacer moiety comprised of one or more atoms;

(b′) is zero or an integer having a value of one through ten;

(c) is an integer having a value of one through ten;

R², in each occurrence, is independently H or an organic radical;

R³, in each occurrence, is independently H or an organic radical; and

IL-2 is a residue of an IL-2 moiety.

Still further exemplary conjugates of the invention are encompassedwithin the following structure:

wherein:

each (n) is independently an integer having a value of from 2 to 4000;and

IL-2 is a residue of IL-2 moiety.

Exemplary conjugates that include a releasable linkage include those inwhich an IL-2 moiety are conjugated to a polymeric reagent encompassedwithin the following formula:

wherein:

POLY¹ is a first water-soluble polymer;

POLY² is a second water-soluble polymer;

X¹ is a first spacer moiety;

X² is a second spacer moiety;

H_(α) is an ionizable hydrogen atom;

R¹ is H or an organic radical;

R² is H or an organic radical;

(a) is either zero or one;

(b) is either zero or one;

R^(e1), when present, is a first electron altering group;

R^(e2), when present, is a second electron altering group; and

(FG) is a functional group capable of reacting with an amino group of anactive agent to form a releasable linkage, such as a carbamate linkage.Within this formula, polymeric reagents having the more definedstructure are contemplated:

wherein each of POLY¹, POLY², X¹, X², R¹, R², H_(α) and (FG) is aspreviously defined, and R^(e1) is a first electron altering group; andW² is a second electron altering group.

Still further exemplary polymeric reagents fall within the followingformulae:

wherein, for each structure and in each instance, (n) is independentlyan integer from 4 to 1500.

These releasable linkage-providing polymeric reagents can be prepared inaccordance with the procedures set forth in U.S. Patent ApplicationPublication No. 2006/0293499.

Exemplary conjugates formed using releasable linkage-providing polymericreagents include those of the following formulae:

wherein:

POLY¹ is a first water-soluble polymer;

POLY² is a second water-soluble polymer;

X¹ is a first spacer moiety;

X² is a second spacer moiety;

H_(α) is an ionizable hydrogen atom;

R¹ is H or an organic radical;

R² is H or an organic radical;

(a) is either zero or one;

(b) is either zero or one;

R^(e1), when present, is a first electron altering group;

R^(e2), when present, is a second electron altering group;

Y¹ is O or S;

Y² is O or S; and

(IL-2) is a residue of an IL-2 moiety.

Exemplary conjugates have the following structure:

wherein, for each structure and in each instance, (n) is independentlyan integer from 4 to 1500, and (IL-2) is a residue of an IL-2 moiety.

Carboxyl groups represent another functional group that can serve as apoint of attachment on the IL-2 moiety. Structurally, the conjugate willcomprise the following:

where (IL-2) and the adjacent carbonyl group corresponds to thecarboxyl-containing IL-2 moiety, X is a linkage, preferably a heteroatomselected from O, N(H), and S, and POLY is a water-soluble polymer suchas PEG, optionally terminating in an end-capping moiety.

The C(O)—X linkage results from the reaction between a polymericderivative bearing a terminal functional group and a carboxyl-containingIL-2 moiety. As discussed above, the specific linkage will depend on thetype of functional group utilized. If the polymer is end-functionalizedor “activated” with a hydroxyl group, the resulting linkage will be acarboxylic acid ester and X will be O. If the polymer backbone isfunctionalized with a thiol group, the resulting linkage will be athioester and X will be S. When certain multi-arm, branched or forkedpolymers are employed, the C(O)X moiety, and in particular the X moiety,may be relatively more complex and may include a longer linkagestructure.

Water-soluble derivatives containing a hydrazide moiety are also usefulfor conjugation at a carbonyl and carboxylic acid. To the extent thatthe IL-2 moiety does not contain a carbonyl moiety or a carboxylic acid,one can be added using techniques known to one of ordinary skill in theart. For example, a carbonyl moiety can be introduced by reducing acarboxylic acid (e.g., the C-terminal carboxylic acid) and/or byproviding glycosylated or glycated (wherein the added sugars have acarbonyl moiety) versions of the IL-2 moiety. With respect to IL-2moieties containing a carboxylic acid, a PEG-hydrazine reagent can, inthe presence of a coupling agent (e.g., DCC), covalently attach to theIL-2 moiety [e.g., mPEG-OCH₂C(O)NHNH₂+HOC(O)-(IL-2) results inmPEG-OCH₂C(O)NHNHC(O)-IL-2]. Specific examples of water-solublederivatives containing a hydrazide moiety, along with the correspondingconjugates, are provided in Table 2, below. In addition, anywater-soluble derivative containing an activated ester (e.g., asuccinimidyl group) can be converted to contain a hydrazide moiety byreacting the water-soluble polymer derivative containing the activatedester with hydrazine (NH₂—NH₂) or tert-butyl carbazate[NH₂NHCO₂C(CH₃)₃]. In the table, the variable (n) represents the numberof repeating monomeric units and “—C(O)-(IL-2)” represents the residueof the IL-2 moiety following conjugation to the polymeric reagent.Optionally, the hydrazone linkage can be reduced using a suitablereducing agent. While each polymeric portion [e.g., (OCH₂CH₂)_(n) or(CH₂CH₂O)_(n)] presented in Table 2 terminates in a “CH₃” group, othergroups (such as H and benzyl) can be substituted therefor.

TABLE 2 Carboxyl-Specific Polymeric Reagents and the IL-2 MoietyConjugate Formed Therefrom Polymeric Reagent Corresponding Conjugate

mPEG-Hydrazine Reagents Hydrazone Linkage

mPEG-Hydrazine Reagents Hydrazone Linkage

mPEG-Hydrazine Reagents Hydrazone Linkage

mPEG-Hydrazine Reagents Hydrazone Linkage

mPEG-Hydrazine Reagents Hydrazone Linkage

mPEG-Hydrazine Reagents Hydrazone Linkage

mPEG-Hydrazine Reagents Hydrazone Linkage

mPEG-Hydrazine Reagents Hydrazone Linkage

mPEG-Hydrazine Reagents C(O)NHNHC(O) Linkage

Thiol groups contained within the IL-2 moiety can serve as effectivesites of attachment for the water-soluble polymer. In particular,cysteine residues provide thiol groups when the IL-2 moiety is aprotein. The thiol groups in such cysteine residues can then be reactedwith an activated PEG that is specific for reaction with thiol groups,e.g., an N-maleimidyl polymer or other derivative as described in U.S.Pat. No. 5,739,208 and in WO 01/62827. In addition, a protected thiolmay be incorporated into an oligosaccharide side chain of an activatedglycoprotein, followed by deprotection with a thiol-reactivewater-soluble polymer.

Specific examples of reagents, along with the corresponding conjugate,are provided in Table 3, below. In the table, the variable (n)represents the number of repeating monomeric units and “—S-(IL-2)”represents the IL-2 moiety residue following conjugation to thewater-soluble polymer. While each polymeric portion [e.g., (OCH₂CH₂)_(n)or (CH₂CH₂O)_(n)] presented in Table 3 terminates in a “CH₃” group,other groups (such as H and benzyl) can be substituted therefor.

With respect to SEQ ID NOs: 1 and 2 corresponding to exemplary IL-2moieties, it can be seen that there is a cysteine residue at position125. Thus, an exemplary thiol attachment sites is the cysteine locatedat position 125. Although it is preferred not to disrupt any disulfidebonds, associated with a given IL-2 moiety, it may be possible to attacha polymer within the side chain of one or more of these cysteineresidues and retain a degree of activity. In addition, it is possible toadd a cysteine residue to the IL-2 moiety using conventional synthetictechniques. See, for example, the procedure described in WO 90/12874 foradding cysteine residues, wherein such procedure can be adapted for anIL-2 moiety. In addition, conventional genetic engineering processes canalso be used to introduce a cysteine residue into the IL-2 moiety. Insome embodiments, however, it is preferred not to introduce anadditional cysteine residue and/or thiol group.

TABLE 3 Thiol-Selective Polymeric Reagents and the IL-2 Moiety ConjugateFormed Therefrom Polymeric Reagent Corresponding Conjugate

mPEG Maleimide Reagent Thioether Linkage

mPEG Maleimide Reagent Thioether Language

mPEG Maleimide Reagent Thioether Linkage

Homobifunctional mPEG Maleimide Reagent Thioether Linkages

mPEG Maleimide Reagent Thioether Linkage

Thioether Linkage mPEG Maleimide Reagent

Thioether Linkage mPEG Maleimide Reagent

Thioether Linkage mPEG Forked Maleimide Reagent

branched mPEG2 Maleimide Reagent Thioether Linkage

branched mPEG2 Maleimide Reagent Thioether Linkage

Branched mPEG2 Forked Maleimide Reagent

Thioether Linkages

Branched mPEG2 Forked Maleimide Reagent Thioether Linkages

mPEG Vinyl Sulfone Reagent Thioether Linkage

mPEG Thiol Reagent Disulfide Linkage

Homobifunctional PEG Thiol Reagent Disulfide Linkages

H₃CO—(CH₂CH₂O)_(n)—CH₂CH₂CH₂CH₂—S—S—(IL-2) Disulfide Linkage mPEGDisulfide Reagent

(IL-2)—S—S—CH₂CH₂—(CH₂CH₂O)_(n)—CH₂CH₂CH₂CH₂—S—S—(IL-2) DisulfideLinkages Homobifunctional Disulfide Reagent

With respect to conjugates formed from water-soluble polymers bearingone or more maleimide functional groups (regardless of whether themaleimide reacts with an amine or thiol group on the IL-2 moiety), thecorresponding maleamic acid form(s) of the water-soluble polymer canalso react with the IL-2 moiety. Under certain conditions (e.g., a pH ofabout 7-9 and in the presence of water), the maleimide ring will “open”to form the corresponding maleamic acid. The maleamic acid, in turn, canreact with an amine or thiol group of an IL-2 moiety. Exemplary maleamicacid-based reactions are schematically shown below. POLY represents thewater-soluble polymer, and (IL-2) represents the IL-2 moiety.

A representative conjugate in accordance with the invention can have thefollowing structure:POLY-L_(0,1)-C(O)Z—Y—S—S-(IL-2)wherein POLY is a water-soluble polymer, L is an optional linker, Z is aheteroatom selected from the group consisting of O, NH, and S, and Y isselected from the group consisting of C₂₋₁₀ alkyl, C₂₋₁₀ substitutedalkyl, aryl, and substituted aryl, and (IL-2) is an IL-2 moiety.Polymeric reagents that can be reacted with an IL-2 moiety and result inthis type of conjugate are described in U.S. Patent ApplicationPublication No. 2005/0014903.

As previously indicated, exemplary conjugates of the invention whereinthe water-soluble polymer is in a branched form, will have the branchedform of the water-soluble polymer comprise the following structure:

wherein each (n) is independently an integer having a value of from 2 to4000.

Exemplary conjugates having a water-soluble polymer in branched form areprepared using the following reagent:

thereby forming a conjugate having the following structure:

wherein:

(for each structure) each (n) is independently an integer having a valueof from 2 to 4000; and

IL-2 is a residue of IL-2 moiety.

An additional exemplary conjugate can be formed using a reagent:

thereby forming a conjugate having the following structure:

wherein:

(for each structure) (n) is independently an integer having a value offrom 2 to 4000; and

IL-2 is a residue of IL-2 moiety.

Conjugates can be formed using thiol-selective polymeric reagents in anumber of ways and the invention is not limited in this regard. Forexample, the IL-2 moiety—optionally in a suitable buffer (includingamine-containing buffers, if desired)—is placed in an aqueous media at apH of about 7-8 and the thiol-selective polymeric reagent is added at amolar excess. The reaction is allowed to proceed for about 0.5 to 2hours, although reaction times of greater than 2 hours (e.g., 5 hours,10 hours, 12 hours, and 24 hours) can be useful if PEGylation yields aredetermined to be relatively low. Exemplary polymeric reagents that canbe used in this approach are polymeric reagents bearing a reactive groupselected from the group consisting of maleimide, sulfone (e.g., vinylsulfone), and thiol (e.g., functionalized thiols such as an orthopyridinyl or “OPSS”).

With respect to polymeric reagents, those described here and elsewherecan be purchased from commercial sources or prepared from commerciallyavailable starting materials. In addition, methods for preparing thepolymeric reagents are described in the literature.

The attachment between the IL-2 moiety and the non-peptidicwater-soluble polymer can be direct, wherein no intervening atoms arelocated between the IL-2 moiety and the polymer, or indirect, whereinone or more atoms are located between the IL-2 moiety and the polymer.With respect to the indirect attachment, a “spacer moiety” serves as alinker between the residue of the IL-2 moiety and the water-solublepolymer. The one or more atoms making up the spacer moiety can includeone or more of carbon atoms, nitrogen atoms, sulfur atoms, oxygen atoms,and combinations thereof. The spacer moiety can comprise an amide,secondary amine, carbamate, thioether, and/or disulfide group.Nonlimiting examples of specific spacer moieties include those selectedfrom the group consisting of —O—, —S—, —S—S—, —C(O)—, —C(O)—NH—,—NH—C(O)—NH—, —O—C(O)—NH—, —C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—, —O—CH₂—, —CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—,—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—, —CH₂—O—CH₂—CH₂—CH₂—,—CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—CH₂—O—,—C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—,—CH₂—CH₂—CH₂—CH₂—C(O)—NH—, —C(O)—O—CH₂—, —CH₂—C(O)—O—CH₂—,—CH₂—CH₂—C(O)—O—CH₂—, —C(O)—O—CH₂—CH₂—, —NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—CH₂—, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—CH₂—,—O—C(O)—NH—[CH₂]_(h)—(OCH₂CH₂)_(j)—, bivalent cycloalkyl group, —O—,—S—, an amino acid, —N(R⁶)—, and combinations of two or more of any ofthe foregoing, wherein R⁶ is H or an organic radical selected from thegroup consisting of alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkynyl, substituted alkynyl, aryl and substituted aryl, (h) iszero to six, and (j) is zero to 20. Other specific spacer moieties havethe following structures: —C(O)—NH—(CH₂)₁₋₆—NH—C(O)-,—NH—C(O)—NH—(CH₂)₁₋₆—NH—C(O)-, and —O—C(O)—NH—(CH₂)₁₋₆—NH—C(O)-, whereinthe subscript values following each methylene indicate the number ofmethylenes contained in the structure, e.g., (CH₂)₁₋₆ means that thestructure can contain 1, 2, 3, 4, 5 or 6 methylenes. Additionally, anyof the above spacer moieties may further include an ethylene oxideoligomer chain comprising 1 to 20 ethylene oxide monomer units [i.e.,—(CH₂CH₂O)₁₋₂₀]. That is, the ethylene oxide oligomer chain can occurbefore or after the spacer moiety, and optionally in between any twoatoms of a spacer moiety comprised of two or more atoms. Also, theoligomer chain would not be considered part of the spacer moiety if theoligomer is adjacent to a polymer segment and merely represent anextension of the polymer segment.

Compositions

The conjugates are typically part of a composition. Generally, thecomposition comprises a plurality of conjugates, preferably although notnecessarily, each conjugate is comprised of the same IL-2 moiety (i.e.,within the entire composition, only one type of IL-2 moiety is found).In addition, the composition can comprise a plurality of conjugateswherein any given conjugate is comprised of a moiety selected from thegroup consisting of two or more different IL-2 moieties (i.e., withinthe entire composition, two or more different IL-2 moieties are found).Optimally, however, substantially all conjugates in the composition(e.g., 85% or more of the plurality of conjugates in the composition)are each comprised of the same IL-2 moiety.

The composition can comprise a single conjugate species (e.g., amonoPEGylated conjugate wherein the single polymer is attached at thesame location for substantially all conjugates in the composition) or amixture of conjugate species (e.g., a mixture of monoPEGylatedconjugates where attachment of the polymer occurs at different sitesand/or a mixture monPEGylated, diPEGylated and triPEGylated conjugates).The compositions can also comprise other conjugates having four, five,six, seven, eight or more polymers attached to any given moiety havingIL-2 activity. In addition, the invention includes instances wherein thecomposition comprises a plurality of conjugates, each conjugatecomprising one water-soluble polymer covalently attached to one IL-2moiety, as well as compositions comprising two, three, four, five, six,seven, eight, or more water-soluble polymers covalently attached to oneIL-2 moiety.

With respect to the conjugates in the composition, the composition willsatisfy one or more of the following characteristics at least about 85%of the conjugates in the composition will have from one to four polymersattached to the IL-2 moiety; at least about 85% of the conjugates in thecomposition will have from one to three polymers attached to the IL-2moiety; at least about 85% of the conjugates in the composition willhave from one to two polymers attached to the IL-2 moiety; at leastabout 85% of the conjugates in the composition will have one polymerattached to the IL-2 moiety; at least about 95% of the conjugates in thecomposition will have from one to five polymers attached to the IL-2moiety; at least about 95% of the conjugates in the composition willhave from one to four polymers attached to the IL-2 moiety; at leastabout 95% of the conjugates in the composition will have from one tothree polymers attached to the IL-2 moiety; at least about 95% of theconjugates in the composition will have from one to two polymersattached to the IL-2 moiety; at least about 95% of the conjugates in thecomposition will have one polymer attached to the IL-2 moiety; at leastabout 99% of the conjugates in the composition will have from one tofive polymers attached to the IL-2 moiety; at least about 99% of theconjugates in the composition will have from one to four polymersattached to the IL-2 moiety; at least about 99% of the conjugates in thecomposition will have from one to three polymers attached to the IL-2moiety; at least about 99% of the conjugates in the composition willhave from one to two polymers attached to the IL-2 moiety; and at leastabout 99% of the conjugates in the composition will have one polymerattached to the IL-2 moiety. It is understood that a reference to arange of polymers, e.g., “from x toy polymers,” contemplates a number ofpolymers x toy inclusive (that is, for example, “from one to threepolymers” contemplates one polymer, two polymers and three polymers,“from one to two polymers” contemplates one polymer and two polymers,and so forth).

In one or more embodiments, it is preferred that theconjugate-containing composition is free or substantially free ofalbumin. It is also preferred that the composition is free orsubstantially free of proteins that do not have IL-2 activity. Thus, itis preferred that the composition is 85%, more preferably 95%, and mostpreferably 99% free of albumin. Additionally, it is preferred that thecomposition is 85%, more preferably 95%, and most preferably 99% free ofany protein that does not have IL-2 activity. To the extent that albuminis present in the composition, exemplary compositions of the inventionare substantially free of conjugates comprising a poly(ethylene glycol)polymer linking a residue of an IL-2 moiety to albumin.

In the PROLEUKIN® brand of aldesleukin (available from PrometheusLaboratories Inc., San Diego Calif.), IL-2 is provided in combinationwith sodium dodecyl sulfate (“SDS”). In contrast, the compositions ofthe present invention advantageously may not require SDS and aretherefore free (or substantially) free of SDS as well as detergentsgenerally (e.g., Tween 20 and Tween 80). Consequently, the compositionsand conjugates of the present invention can be prepared withoutperforming a step of adding SDS, TWEEN® 20, and TWEEN® 80. In addition,the compositions and conjugates of the present invention can be preparedwithout performing the step of adding a detergent or other excipient.Furthermore, the compositions of the present invention are free orsubstantially free (e.g., less than about 20%, more preferably less thanabout 15%, still more preferably less than about 10%, yet still morepreferably less than about 9%, yet still more preferably less than about8%, yet still more preferably less than about 7%, yet still morepreferably less than about 6%, yet still more preferably less than about5%, yet still more preferably less than about 4%, yet still morepreferably less than about 3%, yet still more preferably less than about2%, yet still more preferably less than about 1%, yet still morepreferably less than about 0.5%, with less than 0.001% being mostpreferred) of detergents such as SDS, TWEEN® 20, and TWEEN® 80. Inaddition, the compositions and conjugates of the present invention canbe prepared without performing the step of removing (by, for example,ultra-filtration) detergents such as SDS, TWEEN® 20, and TWEEN® 80.Furthermore, the compositions and conjugates of the present inventioncan be prepared without performing the step of removing (by, forexample, ultra-filtration) a detergent.

Control of the desired number of polymers for any given moiety can beachieved by selecting the proper polymeric reagent, the ratio ofpolymeric reagent to the IL-2 moiety, temperature, pH conditions, andother aspects of the conjugation reaction. In addition, reduction orelimination of the undesired conjugates (e.g., those conjugates havingfour or more attached polymers) can be achieved through purificationmeans.

For example, the polymer-IL-2 moiety conjugates can be purified toobtain/isolate different conjugated species. Specifically, the productmixture can be purified to obtain an average of anywhere from one, two,three, four, five or more PEGs per IL-2 moiety, typically one, two orthree PEGs per IL-2 moiety. The strategy for purification of the finalconjugate reaction mixture will depend upon a number of factors,including, for example, the molecular weight of the polymeric reagentemployed, the particular IL-2 moiety, the desired dosing regimen, andthe residual activity and in vivo properties of the individualconjugate(s).

If desired, conjugates having different molecular weights can beisolated using gel filtration chromatography and/or ion exchangechromatography. That is to say, gel filtration chromatography is used tofractionate differently numbered polymer-to-IL-2 moiety ratios (e.g.,1-mer, 2-mer, 3-mer, and so forth, wherein “1-mer” indicates 1 polymerto IL-2 moiety, “2-mer” indicates two polymers to IL-2 moiety, and soon) on the basis of their differing molecular weights (where thedifference corresponds essentially to the average molecular weight ofthe water-soluble polymer portion). For example, in an exemplaryreaction where a 35,000 Dalton protein is randomly conjugated to apolymeric reagent having a molecular weight of about 20,000 Daltons, theresulting reaction mixture may contain unmodified protein (having amolecular weight of about 35,000 Daltons), monoPEGylated protein (havinga molecular weight of about 55,000 Daltons), diPEGylated protein (havinga molecular weight of about 75,000 Daltons), and so forth.

While this approach can be used to separate PEG and other polymer-IL-2moiety conjugates having different molecular weights, this approach isgenerally ineffective for separating positional isoforms havingdifferent polymer attachment sites within the IL-2 moiety. For example,gel filtration chromatography can be used to separate from each othermixtures of PEG 1-mers, 2-mers, 3-mers, and so forth, although each ofthe recovered conjugate compositions may contain PEG(s) attached todifferent reactive groups (e.g., lysine residues) within the IL-2moiety.

Gel filtration columns suitable for carrying out this type of separationinclude SUPERDEX™ and SEPHADEX™ columns available from AmershamBiosciences (Piscataway, N.J.). Selection of a particular column willdepend upon the desired fractionation range desired. Elution isgenerally carried out using a suitable buffer, such as phosphate,acetate, or the like. The collected fractions may be analyzed by anumber of different methods, for example, (i) absorbance at 280 nm forprotein content, (ii) dye-based protein analysis using bovine serumalbumin (BSA) as a standard, (iii) iodine testing for PEG content (Simset al. (1980) Anal. BioIL-2m, 107:60-63), (iv) sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS PAGE), followed by staining withbarium iodide, and (v) high performance liquid chromatography (HPLC).

Separation of positional isoforms is carried out by reverse phasechromatography using a reverse phase-high performance liquidchromatography (RP-HPLC) using a suitable column (e.g., a C₁₈ column orC3 column, available commercially from companies such as AmershamBiosciences or Vydac) or by ion exchange chromatography using an ionexchange column, e.g., a SEPHAROSE® ion exchange column available fromAmersham Biosciences. Either approach can be used to separatepolymer-active agent isomers having the same molecular weight (i.e.,positional isoforms).

The compositions are preferably substantially free of proteins that donot have IL-2 activity. In addition, the compositions preferably aresubstantially free of all other noncovalently attached water-solublepolymers. In some circumstances, however, the composition can contain amixture of polymer-IL-2 moiety conjugates and unconjugated IL-2 moiety.

Optionally, the composition of the invention further comprises apharmaceutically acceptable excipient. If desired, the pharmaceuticallyacceptable excipient can be added to a conjugate to form a composition.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, amino acids,and combinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, cyclodextrins, andthe like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The composition can also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for one or more embodiments of the present inventioninclude benzalkonium chloride, benzethonium chloride, benzyl alcohol,cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol,phenylmercuric nitrate, thimersol, and combinations thereof.

An antioxidant can be present in the composition as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in one or more embodiments of the present inventioninclude, for example, ascorbyl palmitate, butylated hydroxyanisole,butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propylgallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodiummetabisulfite, and combinations thereof.

A surfactant can be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as TWEEN® 20 and TWEEN® 80, and PLURONIC®surfactants such as PLURONIC® F-68 and PLURONIC® F-88 (both of which areavailable from BASF, Mount Olive, N.J.); sorbitan esters; lipids, suchas phospholipids such as lecithin and other phosphatidylcholines,phosphatidylethanolamines (although preferably not in liposomal form),fatty acids and fatty esters; steroids, such as cholesterol; and EDTA,zinc and other such suitable cations.

Acids or bases can be present as an excipient in the composition.Nonlimiting examples of acids that can be used include those acidsselected from the group consisting of hydrochloric acid, acetic acid,phosphoric acid, citric acid, malic acid, lactic acid, formic acid,trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid,sulfuric acid, fumaric acid, and combinations thereof. Examples ofsuitable bases include, without limitation, bases selected from thegroup consisting of sodium hydroxide, sodium acetate, ammoniumhydroxide, potassium hydroxide, ammonium acetate, potassium acetate,sodium phosphate, potassium phosphate, sodium citrate, sodium formate,sodium sulfate, potassium sulfate, potassium fumerate, and combinationsthereof.

One or more amino acids can be present as an excipient in thecompositions described herein. Exemplary amino acids in this regardinclude arginine, lysine and glycine.

The amount of the conjugate (i.e., the conjugate formed between theactive agent and the polymeric reagent) in the composition will varydepending on a number of factors, but will optimally be atherapeutically effective dose when the composition is stored in a unitdose container (e.g., a vial). In addition, the pharmaceuticalpreparation can be housed in a syringe. A therapeutically effective dosecan be determined experimentally by repeated administration ofincreasing amounts of the conjugate in order to determine which amountproduces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, the excipient will be present in the composition inan amount of about 1% to about 99% by weight, preferably from about 5%to about 98% by weight, more preferably from about 15 to about 95% byweight of the excipient, with concentrations less than 30% by weightmost preferred.

These foregoing pharmaceutical excipients along with other excipientsare described in “Remington: The Science & Practice of Pharmacy”, 19thed., Williams & Williams, (1995), the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H.,Handbook of Pharmaceutical Excipients, 3rd Edition, AmericanPharmaceutical Association, Washington, D.C., 2000.

The compositions encompass all types of formulations and in particularthose that are suited for injection, e.g., powders or lyophilates thatcan be reconstituted as well as liquids. Examples of suitable diluentsfor reconstituting solid compositions prior to injection includebacteriostatic water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline, sterile water,deionized water, and combinations thereof. With respect to liquidpharmaceutical compositions, solutions and suspensions are envisioned.

The compositions of one or more embodiments of the present invention aretypically, although not necessarily, administered via injection and aretherefore generally liquid solutions or suspensions immediately prior toadministration. The pharmaceutical preparation can also take other formssuch as syrups, creams, ointments, tablets, powders, and the like. Othermodes of administration are also included, such as pulmonary, rectal,transdermal, transmucosal, oral, intrathecal, intratumorally,peritumorally, intraperitonally, subcutaneous, intra-arterial, and soforth.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with conjugate. The method comprisesadministering to a patient, generally via injection, a therapeuticallyeffective amount of the conjugate (preferably provided as part of apharmaceutical composition). As previously described, the conjugates canbe injected (e.g., intramuscularly, subcutaneously and parenterally).Suitable formulation types for parenteral administration includeready-for-injection solutions, dry powders for combination with asolvent prior to use, suspensions ready for injection, dry insolublecompositions for combination with a vehicle prior to use, and emulsionsand liquid concentrates for dilution prior to administration, amongothers.

The method of administering the conjugate (preferably provides as partof a pharmaceutical composition) can optionally be conducted so as tolocalize the conjugate to a specific area. For example, the liquid, geland solid formulations comprising the conjugate could be surgicallyimplanted in a diseased area (such as in a tumor, near a tumor, in aninflamed area, and near an inflamed area). Conveniently, organs andtissue can also be imaged in order to ensure the desired location isbetter exposed to the conjugate.

The method of administering may be used to treat any condition that canbe remedied or prevented by administration of the conjugate. Those ofordinary skill in the art appreciate which conditions a specificconjugate can effectively treat. For example, the conjugates can be usedeither alone or in combination with other pharmacotherapy to treatpatients suffering from a malady selected from the group consisting ofrenal cell carcinoma, metastatic melanoma, hepatitis C virus (HCV),human immunodeficiency virus (HIV), acute myeloid leukemia,non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, juvenile rheumatoidarthritis, atopic dermatitis, breast cancer and bladder cancer.Advantageously, the conjugate can be administered to the patient priorto, simultaneously with, or after administration of another activeagent.

The actual dose to be administered will vary depending upon the age,weight, and general condition of the subject as well as the severity ofthe condition being treated, the judgment of the health careprofessional, and conjugate being administered. Therapeuticallyeffective amounts are known to those skilled in the art and/or aredescribed in the pertinent reference texts and literature. Generally, atherapeutically effective amount will range from about 0.001 mg to 100mg, preferably in doses from 0.01 mg/day to 75 mg/day, and morepreferably in doses from 0.10 mg/day to 50 mg/day. A given dose can beperiodically administered up until, for example, symptoms oforganophosphate poisoning lessen and/or are eliminated entirely.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

It is to be understood that while the invention has been described inconjunction with the preferred specific embodiments thereof, that theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All articles, books, patents and other publications referenced hereinare hereby incorporated by reference in their entireties.

EXPERIMENTAL

The practice of the invention will employ, unless otherwise indicated,conventional techniques of organic synthesis, biochemistry, proteinpurification and the like, which are within the skill of the art. Suchtechniques are fully explained in the literature. See, for example, J.March, Advanced Organic Chemistry: Reactions Mechanisms and Structure,4th Ed. (New York: Wiley-Interscience, 1992), supra.

In the following examples, efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperatures, etc.) butsome experimental error and deviation should be taken into account.Unless indicated otherwise, temperature is in degrees C. and pressure isat or near atmospheric pressure at sea level. Each of the followingexamples is considered to be instructive to one of ordinary skill in theart for carrying out one or more of the embodiments described herein.

An aqueous solution (“stock solution”) comprising recombinant IL-2(“rIL-2”) corresponding to the amino acid sequence of SEQ ID NO: 3, themature protein sequence was obtained from Myoderm (Norristown Pa.) foruse in the examples or was prepared in accordance with Example 1. Theconcentration of the stock solution varied between 1 and 100 mg/mL.

SDS-PAGE Analysis

Samples were analyzed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) using the Invitrogen NuUPAGE® system andNovex 4-10% Bis-Tris pre-cast gels (Invitrogen, Carlsbad, Calif.).Samples were prepared, loaded on the gel and electrophoresis performedas described by the manufacturer.

Cation Exchange Chromatography

A SP-HP SEPHAROSE® (GE Healthcare) cation exchange column with a bedvolume of approximately 100 ml was prepared using standard methods. Thecolumn was connected to a GE Healthcare (Chalfont St. Giles, UK)AKTAexplorer™100 to purify the prepared PEG-rIL-2 conjugates. Detailsfor the purification process are described below.

RP-HPLC Analysis

Reversed-phase chromatography (RP-HPLC) analysis was performed on anAgilent (Santa Clara, Calif.) 1100 HPLC system. Samples were analyzedusing a Silverton (Japan) Intrada WP-RP column (3 um particle size,2.1×150 mm). The flow rate of the column was 0.5 ml/min. The mobilephases were 0.09% TFA in water (solvent A) and 0.04% TFA in acetonitrile(solvent B).

Example 1 Cloning of the IL-2 Gene and Expression of rIL-2

The human IL-2 cDNA sequence may not be optimally expressed inprokaryotes like E. coli due to significant differences in the codonusage of the different organisms. Instead of doing many point mutationsto the existing human-derived cDNA sequence to maximize E. coli codonusage, a PCR technique was employed to fully synthesize the gene.

The method for synthesizing the gene from overlapping primers wasessentially a combination of two methods with minor modifications. Abasic discussion of each individual method is provided in Young et al.(2004) Nucleic Acids Research 32(7):e59 and Devlin et al. (1988) Gene65:13-22. Briefly, the DNA sequence was divided into forward and reverseoligonucleotides of less than 35 bp with a few exceptions and there wereno gaps between the oligonucleotides. Each oligonucleotide overlappedthe two adjacent ones on the opposite strand by at least 10 nucleotidesat the 3′ ends and at least 15 nucleotides at the 5′ ends. Dualasymmetric PCR was used to assemble sub-fragments of the gene and thesewere combined to assemble the entire gene using overlap extension PCR. AT7 endonuclease I selection step was then used to remove mismatchedduplexes as described by Young et. al. See Young et al. (2004) NucleicAcids Research 32(7):e59. Restriction enzyme sites were included at thegene termini and the final gene fragment was cloned into a commerciallyavailable expression vector for E. coli. DNA sequence analysis was usedto confirm the sequence obtained as shown in FIG. 1 and SEQ ID NO: 5.

Using this approach, the amino acid sequence excludes amino acidposition #1 (alanine) as compared to the native mature human sequenceand includes a C→S amino acid mutation at amino acid position #125 forthe sequence as shown. The first amino acid in this sequence is amethionine for direct bacterial expression (no signal peptide encoded).Upon expression, however, the initial methionine is removed by hostmethionine amino peptidase.

The gene was cloned into one of the pET (T7) expression vectors. Theprotein was expressed in the E. coli strain BL21(DE3), one of thestrains typically used for the T7 expression system. This expressionsystem is available commercially and the methods for expression areavailable from EMD Biosciences, Merck KGaA, Darmstadt, Germany. The useof this system was done under a research license from the BrookhavenNational Laboratory. The vector resulted in the protein being expressedas inclusion bodies in E. coli. Typical recipes used for expression canbe found in the literature and in Protein Production by Auto-Inductionin High-Density Shaking Cultures, by F. William Studier, BiologyDepartment, Brookhaven National Laboratory, Upton, N.Y. 11973 (Dec. 20,2007).

Following fermentation, the cells were harvested by centrifugation. Thecell mass pellet was stored at −80° C. for future homogenization. Thefrozen cell mass pellet was re-suspended in cell wash buffer (50 mMTris, 5 mM EDTA, pH8.0) to a concentration of 10% (WN) and centrifugedat 13860×g for 30 minutes. The supernatant was discarded. The washedpellet was re-suspended in homogenization buffer (50 mM Tris, 5 mM EDTA,1 mM PMSF, pH8.0) and homogenized by a Microfluidizer (M-110P fromMicrofluidics, Newton, Mass., USA) at 4-15° C. for one pass. Thehomogenate was diluted 2-fold using cell wash buffer (50 mM Tris, 5 mMEDTA, pH8.0) and centrifuged at 13860×g for 60 minutes. The supernatantwas discarded. The inclusion body pellet was washed in three steps usingbuffers sequentially of 50 mM Tris, 5 mM EDTA, 2% TRITON® X-100, pH8.0;50 mM Tris, 5 mM EDTA, 1% sodium deoxycholate, pH8.0; and 50 mM Tris, 5mM EDTA, 1M NaCl, pH8.0. After washing, the crude IL-2 inclusion bodieswere obtained.

The crude IL-2 inclusion bodies were dissolved into 6M guanidine, 100 mMTris, pH8 buffer. EDTA was added to final concentration 2 mM.Dithiothreitol (DTT) was then added to final concentration 50 mM. Themixture was incubated at 50° C. for 30 minutes. After reduction, waterwas added to the mixture to reduce guanidine concentration to 4.8. Afterone hour of centrifuging at 13860×g, the resulting gel-like pellet wasdiscarded. The guanidine concentration in the supernatant was furtherreduced to 3.5M by adding water. The pH was adjusted to 5 with titrationof 100% acetic acid. The mixture was incubated at room temperature for60 minutes and centrifuged at 13860×g for one hour. The resulting pelletwas suspended into 3.5M guanidine, 20 mM acetate, 5 mM DTT, pH 5 bufferand centrifuged at 13860×g for one hour. This washing step was repeatedone more time.

The clean and reduced IL-2 inclusion bodies were dissolved into 6Mguanidine, 100 mM Tris pH8 buffer. 100 mM CuCl₂ stock was added to reacha final Cu²⁺ concentration 0.1 mM. The mixture was incubated at 4′Covernight.

Another embodiment of the invention is directed to an improved method ofallowing proteins to obtain tertiary structure. In this regard, previousmethods often relied upon step dilution, which is often harsh toproteins. Thus, in an improved approach to allow for the folding ofproteins under more gentle conditions, a method is provided wherein themethod comprises the step of placing an expressed protein (e.g., an IL-2moiety, such as IL-2 prepared in accordance with this example) within adialysis bag having a pore size less than the size of the expressedprotein, and adding (preferably over several hours, e.g., over 6 hours,more preferably over 10 hours, and still more preferably over 15 hours)a protein denaturant-free solution (e.g., water). Exemplary proteindenaturant-free solutions are recognized to those of ordinary skill inthe art and include, for example, solutions (e.g., buffers and water)that lack (or substantially lack) guanidine, urea, lithium perchlorate,2-mercaptoethanol, dithiothreitol and detergents. Thus, in accordancewith this method, the expressed IL-2 solution was put into dialysis bags(having a molecular weight pore size of 3.5 kiloDaltons). The dialysisbags were put into a reservoir containing 4.8M guanidine, 0.1M Tris, pH8buffer. After three hours equilibration, the guanidine concentration inthe reservoir was slowly reduced to 2M by pumping water into thereservoir over a period of 15 hours. The entire refolding process wascompleted at 4° C. The refolded IL-2 was checked with SEC-HPLC.

The refolded IL-2 was centrifuged at 13860×g for 60 minutes to removeprecipitates. The supernatant was concentrated with PELLICON® XL TFFmembrane system (Millipore Corporation, USA).

The refolded and concentrated IL-2 was loaded on a BPG column (GEHealthcare Bio-Sciences AB, Uppsala Sweden) packed with SEPHACRYL® S-100HR resin. The running buffer was 2M guanidine, 20 mM Tris pH8 and flowrate was 25 mL/min. The fractions under the IL-2 monomer peak werepooled. It should be noted that other suitable purification methods mayalso be employed, such as ion exchange chromatography and hydrophobicinteraction chromatography (HIC chromatography).

The IL-2 monomer fraction pool was concentrated to about 1-2 mg/mL usingPELLICON® XL TFF membrane system (Millipore Corporation, USA) at 4° C.and 30-40 psi operation pressure. The concentrated IL-2 monomer solutionwas dialyzed into final formulation buffer (10 mM acetate-Na, 5%trehalose, pH 4.5) to bring down the guanidine concentration lower than0.1 mM by changing the formulation buffer several times (4-5 times innormal). The formulated IL-2 solution was rendered sterile by passing a0.22 um filter and stored in −80° C. for further use.

Example 2 PEGylation of rIL-2 with mPEG2-C₂-Fmoc-20K-NHS

mPEG2-C2-Fomc-20K—N-Hydroxysuccinimide Derivative, 20 kDa,(“mPEG2-C2-Fmoc-20K-NHS”)

mPEG2-C2-fmoc-20K-NHS, stored at −80° C. under argon, was warmed toambient temperature under nitrogen purging. A stock solution (200 mG/mL)of mPEG2-C2-fmoc-20K-NHS was prepared in 2 mM HCl, andmPEG2-C2-fmoc-20K-NHS was added to the rIL-2 in an amount sufficient toreach a molar ratio of mPEG2-C2-fmoc-20K-NHS to rIL-2 of 100:1. Thefinal concentration of rIL-2 in the mixture was 0.5 mG/mL (0.035 mM).Sodium bicarbonate buffer (1 M, pH 9.0) was added to the mixture toreach a final concentration of 20 mM, and conjugation was allowed toproceed for thirty minutes to provide [mPEG2-C2-fmoc-20K]-[rIL-2]conjugates. After thirty minutes, quenching was achieved by adding 1 Mglycine (pH 6.0) to the reaction mixture to achieve a finalconcentration of 100 mM. The quenched reaction mixture was then dilutedwith H₂O to provide a conductivity below 0.5 mS/cm (25° C.). The pH wasadjusted to 4.0 using glacial acetic acid prior to column chromatographypurification.

A typical cation exchange chromatography purification profile of[mPEG2-C2-fmoc-20K]-[rIL-2] is provided in FIG. 2.1. The[mPEG2-C2-fmoc-20K]-[rIL-2] and unreacted PEG are indicated and thelines correspond to absorbance at various wavelengths (e.g., 280 nm and225 nm). Purity analysis of [mPEG2-C2-fmoc-20K]-[rIL-2] by reverse phaseHPLC analysis detected purity of the purified conjugate of 100% at 280nm. See FIG. 2.2. Purity was not less than 95% as determined by 4-12%NUPAGE® Bis-Tris SDS-PAGE gel with Coomassie Blue Staining (gel notshown) with 20 μg of the purified [mPEG2-C₂-fmoc-20K]-[rIL-2]. Theapparent large molecular weight of the conjugate, higher than 200 kDa,was believed to be the result of the slow mobility of the conjugatethrough the gel due to a high degree of PEG hydration and resultingrelatively large hydrodynamic radius. Through these tests, it wasconfirmed that three conjugates were produced: a 4-mer, 3-mer, 2-mer and1-mer, i.e., a [mPEG2-C2-fmoc-20K]-[rIL-2] in which four“[mPEG2-C2-fmoc-20K]” are attached to a single “[rIL-2]” for a 4-mer,three “[mPEG2-C2-fmoc-20K]” are attached to a single “[rIL-2]” for a3-mer, two “[mPEG2-C2-fmoc-20K]” are attached to a single [rIL-2] for a2-mer, and one “[mPEG2-C2-fmoc-20K]” attached to a single [rIL-2] for a1-mer.

The releasable nature of [mPEG2-C2-fmoc-20K]-[rIL-2] to liberate rIL-2was shown by detecting the change of species with reverse phase HPLC.Briefly, purified [mPEG2-C2-fmoc-20K]-[rIL-2] was incubated in 100 mMNaHCO₃ solution, at pH 9.0, 37° C., for several hours. Periodically,aliquots of the system were obtained and tested to detect thedisappearance of [mPEG2-C2-fmoc-20K]-[rIL-2] conjugate and the presenceof liberated rIL-2. The appearance of rIL-2 plateaued around ten hoursafter incubation, with a gradual decrease possibly due to precipitation.Data is provided in FIG. 2.3.

Example 3 PEGylation of rIL-2 with mPEG2-CAC-fmoc-20K-NHS

mPEG2-CAC-fmoc-20K—N-Hydroxysuccinimide Derivative, 20 kDa,(“mPEG2-CAC-fmoc-20K-NHS”)

mPEG2-CAC-fmoc-20K-NHS, stored at −80° C. under argon, was warmed toambient temperature under nitrogen purging. A stock solution (200 mG/mL)of mPEG2-CAC-fmoc-20K-NHS was prepared in 2 mM HCl, andmPEG2-CAC-fmoc-20K-NHS was added to the rIL-2 in an amount sufficient toreach a molar ratio of mPEG2-CAC-fmoc-20K-NHS to rIL-2 of 100:1. Thefinal concentration of rIL-2 in the mixture was 0.5 mG/mL (0.035 mM).Sodium bicarbonate buffer (1 M, pH 9.0) was added to the mixture toreach a final concentration of 20 mM, and conjugation was allowed toproceed for thirty minutes to provide [mPEG2-CAC-fmoc-20K]-[rIL-2]conjugates. After thirty minutes, quenching was achieved by adding 1 Mglycine (pH 6.0) to the reaction mixture to achieve a finalconcentration of 100 mM. The quenched reaction mixture was then dilutedwith H₂O to provide a conductivity below 0.5 mS/cm (25° C.). The pH wasadjusted to 4.0 using glacial acetic acid prior to column chromatographypurification.

A typical cation exchange chromatography purification profile of[mPEG2-CAC-fmoc-20K]-[rIL-2] is provided in FIG. 3.1. The[mPEG2-CAC-fmoc-20K]-[rIL-2] is indicated and the lines correspond toabsorbance at various wavelengths. Purity analysis of[mPEG2-CAC-fmoc-20K]-[rIL-2] by reverse phase HPLC analysis detectedpurity of the purified conjugate of 98.5% at 280 nm. The peak at 19.6minutes represents unreacted mPEG2-CAC-fmoc-20K-NHS (which constituted<0.1%). See FIG. 3.2. Purity was not less than 95% as determined by4-12% NUPAGE® Bis-Tris SDS-PAGE gel with Coomassie Blue Staining (gelnot shown) with 20 μg of the purified [mPEG2-CAC-fmoc-20K]-[rIL-2]. Theapparent large molecular weight of the conjugate, higher than 200 kDa,was believed to be the result of the slow mobility of the conjugatethrough the gel due to a high degree of PEG hydration. The molecularweight of purified [mPEG2-CAC-fmoc-20K]-[rIL-2] conjugates was alsodetermined by MALDI-TOF spectrophotometry. As seen in FIG. 3.3, themajor peak at 79.6 kDa is within the expected range for the molecularweight of the 3-mer [mPEG2-CAC-fmoc-20K]-[rIL-2] conjugate. The peak at100.8 kDa is within the expected range for the molecular weight of the4-mer [mPEG2-CAC-fmoc-20K]-[rIL-2]. The peaks with MW 40 kDa and 58.7kDa may represent doubly charged 3-mer IL-2 conjugate and 4-mer IL-2conjugates.

Example 4 PEGylation of rIL-2 with Branched mPEG-N-HydroxysuccinimidylDerivative, 20 kDa

mPEG2-ru-20K—N-Hydroxysuccinimidyl Derivative, 20 kDa,(“mPEG2-ru-20K-NHS”)

mPEG2-ru-20K-NHS, stored at −80° C. under argon, was warmed to ambienttemperature under nitrogen purging. A stock solution (200 mG/mL) ofmPEG2-ru-20K-NHS was prepared in 2 mM HCl, and mPEG2-ru-20K-NHS wasadded to the rIL-2 in an amount sufficient to reach a molar ratio ofmPEG2-ru-20K-NHS to rIL-2 of 100:1. The final concentration of rIL-2 inthe mixture was 0.5 mG/mL (0.035 mM). Sodium bicarbonate buffer (1 M, pH9.0) was added to the mixture to reach a final concentration of 20 mM,and conjugation was allowed to proceed for thirty to provide[mPEG2-ru-20K]-[rIL-2] conjugates. After thirty minutes, quenching wasachieved by adding 1 M glycine (pH 6.0) to the reaction mixture toachieve a final concentration of 100 mM. The quenched reaction mixturewas then diluted with H₂O to provide a conductivity below 0.5 mS/cm (25°C.). The pH was adjusted to 4.0 using glacial acetic acid prior tocolumn chromatography purification.

A typical cation exchange chromatography purification profile of[mPEG2-ru-20K]-[rIL-2] is provided in FIG. 4.1. The[mPEG2-ru-20K]-[rIL-2] and unreacted mPEG2-ru-20K-NHS are indicated andthe lines correspond to absorbance at various wavelengths (e.g., 280 nmand 225 nm). Purity analysis of [mPEG2-ru-20K]-[rIL-2] by reverse phaseHPLC analysis detected purity of the purified conjugate of 100% at 280nm. See FIG. 4.2. Purity was not less than 95% as determined by 4-12%NUPAGE® Bis-Tris SDS-PAGE gel with Coomassie Blue Staining (gel notshown) with 20 μg of purified [mPEG2-ru-20K]-[rIL-2]. The apparent largemolecular weight of the conjugate, higher than 200 kDa, was a result ofthe slow mobility of the conjugate through the gel due to a high degreeof PEG hydration.

Example 5 PEGylation of rIL-2 with Branched mPEG-N-HydroxysuccinimidylDerivative, 40 kDa

mPEG2-ru-40K—N-Hydroxysuccinimidyl Derivative, 40 kDa,(“mPEG2-ru-40K-NHS”)

mPEG2-ru-40K-NHS, stored at −80° C. under argon, was warmed to ambienttemperature under nitrogen purging. A stock solution (200 mG/mL) ofmPEG2-ru-40K-NHS was prepared in 2 mM HCl, and mPEG2-ru-40K-NHS wasadded to the rIL-2 in an amount sufficient to reach a molar ratio ofmPEG2-ru-40K-NHS to rIL-2 of 100:1. The final concentration of rIL-2 inthe mixture was 0.5 mG/mL (0.035 mM). Sodium bicarbonate buffer (1 M, pH9.0) was added to the mixture to reach a final concentration of 20 mM,and conjugation was allowed to proceed for thirty minutes to provide[mPEG2-ru-40K]-[rIL-2] conjugates. After thirty minutes, quenching wasachieved by adding 1 M glycine (pH 4.0) to the reaction mixture toachieve a final concentration of 100 mM. The quenched reaction mixturewas then diluted with H₂O to provide a conductivity below 0.5 mS/cm (25°C.). The pH was adjusted to 4.0 using glacial acetic acid prior tocolumn chromatography purification.

A typical cation exchange chromatography purification profile of[mPEG2-ru-40K]-[rIL-2] is provided in FIG. 5. The [mPEG2-ru-40K]-[rIL-2]and unreacted PEG are indicated and the lines correspond to absorbanceat 280 nm. Purity analysis of [mPEG2-ru-40K]-[rIL-2] by reverse phaseHPLC analysis detected purity of the purified conjugate of 100% at 280nm. Purity was not less than 95% as determined by 4-12% NUPAGE® Bis-TrisSDS-PAGE gel with Coomassie Blue Staining (gel not shown) with 20 μg ofpurified [mPEG2-ru-40K]-[rIL-2]. The apparent large molecular weight ofthe conjugate (likely a 3-mer version of [mPEG2-ru-40K]-[rIL-2]), higherthan 200 kDa, was a result of the slow mobility of the conjugate throughthe gel due to a high degree of PEG hydration. Un-reactedmPEG2-ru-40K-NHS initially eluted through the column followed by[mPEG2-ru-40K]-[rIL-2] conjugates.

Example 6 PEGylation of rIL-2 with Branched mPEG-N-HydroxysuccinimidylDerivative, 4 k Da

mPEG2-ru-20K—N-Hydroxysuccinimidyl Derivative, 4 kDa,(“mPEG2-ru-4K-NHS”)

mPEG2-ru-4K-NHS, stored at −80° C. under argon, was warmed to ambienttemperature under nitrogen purging. A stock solution (200 mG/mL) ofmPEG2-ru-4K-NHS was prepared in 2 mM HCl, and mPEG2-ru-4K-NHS was addedto the rIL-2 in an amount sufficient to reach a molar ratio ofmPEG2-ru-4K-NHS to rIL-2 of 100:1. The final concentration of rIL-2 inthe mixture was 0.5 mG/mL (0.035 mM) solubilized with 0.015% SDS. Sodiumbicarbonate buffer (1 M, pH 9.0) was added to the mixture to reach afinal concentration of 100 mM, and conjugation was allowed to proceedfor thirty minutes to provide [mPEG2-ru-4K]-[rIL-2] conjugates. Afterthirty minutes, quenching was achieved by adding 1 M glycine (pH 4.0) tothe reaction mixture to achieve a final concentration of 100 mM. Thequenched reaction mixture was then diluted with H₂O to provide aconductivity below 0.5 mS/cm (25° C.). The pH was adjusted to 4.0 usingglacial acetic acid prior to column chromatography purification.

A typical cation exchange chromatography purification profile of[mPEG2-ru-4K]-[rIL-2] is provided in FIG. 6. The eluted[mPEG2-ru-4K]-[rIL-2] conjugates showed a mixture of 3-mer, 2-mer and1-mer [mPEG2-ru-4K]-[rIL-2] conjugates in the elution fractions.Fractions containing mixture of 3-/2-mer [mPEG2-ru-4K]-[rIL2], as wellas fractions containing mixture of 2-/1-mer [mPEG2-ru-4K]-[rIL2] werepooled separately, as shown in FIG. 6.

Example 7 PEGylation of rIL-2 with Linear mPEG-Butyraldehyde Derivative,30 kDa

Linear mPEG-Butyraldehyde Derivative, 30 kDa (“mPEG-ButyrALD”)

PEGylation reactions are designed such that after addition of all thereaction components and buffers, the final rIL-2 concentration is 2.5mg/ml. mPEG-ButyrALD, 30 kDa, stored at −20° C. under argon, is warmedto ambient temperature. A quantity of the PEG reagent equal to 10-50 molequivalents of the rIL-2 to be PEGylated is weighed out and dissolved in20 mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12%reagent solution. The 12% PEG reagent solution is added to the aliquotof stock rIL-2 solution and stirred for 15-30 minutes. A reducing agent,sodium cyanoborohydride (NaCNBH₃), is then added at 10-100 molar excessrelative to the PEG reagent and the reaction stirred for 5-18 hours atroom temperature to ensure coupling via a secondary amine linkage tothereby form a conjugate solution.

The aldehyde group of mPEG-ButyrALD is found to react with the primaryamines associated with rIL-2 and covalently bond to them via secondaryamine upon reduction by a reducing reagent such as sodiumcyanoborohydride. Selectivity for which amine(s) become attached withthe polymer can be modulated by adjusting the pH of the conjugationconditions. Relatively low pH conditions (e.g., around a pH of 5.5) willdirect conjugation toward the N-terminus. At relatively neutral pHconditions (e.g., around 7.5 and slightly above), covalent attachmentbecomes more frequent at other locations (i.e., at the amine side chainsof lysine residues contained within the protein). Adjusting the pH ofthe conjugation conditions will allow some degree of control as to whichlocations conjugation occurs, thereby having a better ability to arriveat the desired positional isomers.

Using this same approach, other conjugates are prepared usingmPEG-BuryrALD having other weight average molecular weights.

Example 8 PEGylation of rIL-2 with Branched mPEG-ButyraldehydeDerivative, 40 kDa

Branched mPEG-Butyraldehyde Derivative, 40 kDa (“mPEG2-ButyrALD”)

PEGylation reactions are designed such that after addition of all thereaction components and buffers, the final rIL-2 concentration is 2.5mg/ml. mPEG2-ButyrALD, 40 kDa, stored at −20° C. under argon, is warmedto ambient temperature. A quantity of the PEG reagent equal to 10-50 molequivalents of the rIL-2 to be PEGylated is weighed out and dissolved in20 mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12%reagent solution. The 12% PEG reagent solution is added to the aliquotof stock rIL-2 solution and stirred for 15-30 minutes. A reducing agent,sodium cyanoborohydride (NaCNBH₃), is then added at 10-100 molar excessrelative to the PEG reagent and the reaction stirred for 5-18 hours atroom temperature to ensure coupling via a secondary amine linkage tothereby form a conjugate solution.

The aldehyde group of mPEG2-ButyrALD is found to react with the primaryamines associated with rIL-2 and covalently bond to them via secondaryamine upon reduction by a reducing reagent such as sodiumcyanoborohydride.

Using this same approach, other conjugates are prepared usingmPEG2-BuryrALD having other weight average molecular weights.

Example 9 PEGylation of rIL-2 with Linear mPEG-Succinimidylα-Methylbutanoate Derivative, 30 kDa

Linear mPEG-Succinimidyl α-Methylbutanoate Derivative, 30 kDa(“mPEG-SMB”)

PEGylation reactions are designed such that after addition of all thereaction components and buffers, the final rIL-2 concentration is 2.5mg/ml. mPEG-SMB, 30 kDa, stored at −20° C. under argon, is warmed toambient temperature. A quantity of the PEG reagent equal to 10-50 molequivalents of the rIL-2 to be PEGylated is weighed out and dissolved in20 mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12%reagent solution. The 12% PEG reagent solution is added to the aliquotof stock rIL-2 solution and stirred for 5-18 hours at room temperaturethereby resulting in a conjugate solution. The conjugate solution isquenched with a lysine solution (pH 7.5) such that the final lysinemolar concentration is 10-100 times the PEG reagent molar concentration.

The mPEG-SMB derivative is found to provide a sterically hindered activeNHS ester, which selectively reacts with lysine and terminal amines.

Using this same approach, other conjugates are prepared using mPEG-SMBhaving other weight average molecular weights.

Example 10 PEGylation of rIL-2 with mPEG-PIP, 20 kDa

The basic structure of the polymeric reagent is provided below:

PEGylation reactions are designed such that after addition of all thereaction components and buffers, the final rIL-2 concentration is 2.5mg/ml. mPEG-PIP, 20 kDa, stored at −20° C. under argon, is warmed toambient temperature. A quantity of the PEG reagent equal to 10-50 molequivalents of the rIL-2 to be PEGylated is weighed out and dissolved in20 mM sodium phosphate buffer (pH 7.5) and 1 mM EDTA to form a 12%reagent solution. The 12% PEG reagent solution is added to the aliquotof stock rIL-2 solution and stirred for 15-30 minutes. A reducing agent,sodium cyanoborohydride (NaCNBH₃), is then added at 10-100 molar excessrelative to the PEG reagent and the reaction stirred for 5-18 hours atroom temperature to ensure coupling via a secondary amine linkage (to asecondary carbon) to thereby form a conjugate solution. The conjugatesolution is quenched with a lysine solution (pH 7.5) such that the finallysine molar concentration is 10-100 times the PEG reagent molarconcentration.

The ketone group of mPEG-PIP is found to react with the primary aminesassociated with rIL-2 and covalently bond to them via a secondary amineupon reduction by a reducing reagent such as sodium cyanoborohydride.

Using this same approach, other conjugates are prepared using mPEG-PIPhaving other weight average molecular weights.

Example 11 Activity of Exemplary (rIL-2)-PEG Conjugates

The activity of aldesleukin (control), [mPEG2-C₂-fmoc-20K]-[rIL-2] fromExample 2, [mPEG2-CAC-fmoc-20K]-[rIL-2] from Example 3, and[mPEG2-ru-20K]-[rIL-2] from Example 4 were evaluated in a cellproliferation assay using CTLL-2 cells.

CTLL-2 cells (mouse cytotoxic T lymphocyte cell line) were maintained incomplete RPMI 1640 medium supplemented with 2 mM L-glutamine, 1 mMsodium pyruvate, 10% fetal bovine serum, and 10% IL-2 culture supplement(T-STIM™ with ConA (concanavalin-A)) at 37° C. under a 5% CO₂atmosphere. The cells were cultured in suspension until they reached acell density of 2-3×10⁵ cells/mL before splitting.

For the activity assay, 3-4 days after the last split, the cells werewashed three times in Dulbecco's phosphate buffered saline. The cellswere then re-suspended in supplemented media without T-STIM™ at a celldensity of −2×10⁵ cells/mL and plated in 96-well white walled clearbottom microplates at 90 μl/well. Experiments were also conducted usingsupplemented media (without T-STIM™) adjusted to pH 6.7-7, in order tominimize the release of conjugates during the course of incubation.Then, 10 μl of 10× concentrations of test compound, diluted insupplemented media without T-STIM™, was added. The cells were incubatedat 37° C. in a 5% CO₂ atmosphere for 24 hours. Following the 24 hourincubation, 100 μL of Promega's CELLTITER-GLO® reagent was added to eachwell. The plates were mixed for two minutes on an orbital shaker thenincubated at room temperature for ten minutes. Luminescence was thenrecorded using Perkin Elmer's TOPCOUNT® instrument at an integrationtime of one second/well.

For the [mPEG2-C₂-fmoc-20K]-[rIL-2] releasable conjugates from Example 2and the [mPEG2-CAC-fmoc-20K]-[rIL-2] releasable conjugates from Example3, the activity of both released IL-2 and unreleased conjugates weretested. The test compounds were stored under acidic condition (10 mMsodium acetate buffer, pH 4) to stabilize conjugation. To test theactivity of conjugates, the sample was diluted from the storage bufferinto supplemented media˜one hour prior to the assay. To test theactivity of released IL-2, the releasable conjugates[mPEG2-C2-fmoc-20K]-[rIL-2] conjugates from Example 2 and[mPEG2-CAC-fmoc-20K]-[rIL-2] conjugates from Example 3} were dilutedten-fold in 100 mM (final concentration) sodium bicarbonate buffer, pH 9and pre-incubated at 37° C. for eight hours prior to start of the assay.

The EC₅₀ values (concentration of test compound required to exhibit 50%of maximal response) for cell proliferation were obtained fromnon-linear regression analysis of dose-response curves, using GraphPad'sPrism 5.01 software.

The activities of aldesleukin and the conjugates were measured using acell proliferation assay, and a summary of the results are shown inTable 4. All test articles induced growth of CTLL-2 cells in adose-dependent manner. Since the releasable conjugates werepre-incubated under conditions to force release the protein, aldesleukinwas also pre-incubated as a control to test for stability of the proteinitself under the forced release treatment conditions. As shown in Table4, aldesleukin remained stable following pre-incubation under therelease conditions (8 hours at 37° C., pH 9) and exhibited relativepotency to aldesleukin stored under recommended conditions. Followingpre-incubation of [mPEG2-C2-fmoc-20K]-[rIL-2] from Example 2 and[mPEG2-CAC-fmoc-20K]-[rIL-2] from Example 3 under conditions to inducerelease of IL-2, activity was regained as shown in FIG. 8; IL-2 releasedfrom these conjugates displayed relative potency to the controlaldesleukin, whereas some of the unreleased conjugates were less potentrelative to aldesleukin. The stable 3-mer [mPEG2-ru-20K]-[rIL-2]conjugates displayed the least potency (FIG. 7) and was 0.04% relativeto aldesleukin, but 1-mer [mPEG2-ru-20K]-[rIL-2] showed equivalentpotency to aldesleukin in view the known standard deviation of theassay.

TABLE 4 Summary of CTLL-2 Cell Proliferation in Response to Aldesleukinand PEG-IL-2 conjugates. Potency EC50 Relative to Test compound (ng/mL)Aldesleukin (%) aldesleukin 0.111 102 aldesleukin (release control)0.113 100 3-mer [mPEG2-C2-fmoc-20K]-[rIL-2] 0.076 149 (released) 1-mer[mPEG2-C2-fmoc-20K]-[rIL-2] 0.105 108 (released) 3-mer[mPEG2-CAC-fmoc-20K]- 0.246 46 [rIL-2] (released) 1-mer[mPEG2-CAC-fmoc-20K]- 0.056 202 [rIL-2] (released) 3-mer[mPEG2-C2-fmoc-20K]- 0.497 23 [rIL-2] (unreleased) 1-mer[mPEG2-C2-fmoc-20K]- 0.074 153 [rIL-2] (unreleased) 3-mer[mPEG2-CAC-fmoc-20K]- 5.163 2 [rIL-2] (unreleased) 1-mer[mPEG2-CAC-fmoc-20K]- 0.143 79 [rIL-2] (unreleased) 3-mer[mPEG2-ru-20K]-[rIL-2] 194.400 0.04 1-mer [mPEG2-ru-20K]-[rIL-2] 0.16867

Example 12 Pharmacokinetics of Exemplary (rIL-2)-PEG Conjugates

The pharmacokinetic profiles of aldesleukin (control),[mPEG2-C2-fmoc-20K]-[rIL-2] from Example 2, [mPEG2-CAC-fmoc-20K]-[rIL-2]from Example 3, and [mPEG2-ru-20K]-[rIL-2] from Example 4 were evaluatedin an ELISA following a single injection in mice.

Aldesleukin concentrations were measured by a heterogeneous, sandwichELISA. Briefly, 96-well microtiter plates were coated with mousemonoclonal antibody to IL-2 and blocked. Samples and standard wereprepared in neat plasma and were subsequently diluted to 10% plasma withbuffer containing biotinylated rabbit polyclonal antibodies to IL-2before being incubated on the assay plates. Streptavidin-HorseradishPeroxidase followed by the colorimetric substrate, 3, 3′, 5,5′-tetramethylbenzidine (TMB) was used to detect IL-2. Stop solution wasadded and the absorbance was read at 450 nm with background subtractionat 650 nm. The standard curve was generated by a weighted, 4-parameteralgorithm, and the sample concentrations determined by interpolation tothe standard curve. The lower limit of quantitation was 0.05 ng/mL.

Pooled 1-mer/2-mer [mPEG2-ru-20K]-[rIL-2] concentrations were measuredby a homogeneous HTRF® assay (Cisbio US, Bedford Mass.). Reactionmixture (15 uL, europium chromate conjugated mouse monoclonal antibodyto IL-2, Streptavidin-d2, and biotinylated rabbit monoclonal antibody toPEG) was added to white, low-volume, 384-well microtiter plates. Samplesand standards (5 uL) diluted in neat plasma were added and the platesincubated. The plates were read on a fluorescence reader at 615 and 665nm, and Delta F calculated. The standard curve was generated by aweighted, 5-parameter algorithm and the sample concentrations determinedby interpolation to the standard curve. The lower limit of quatitationwas 0.5 ng/mL.

3-mer [mPEG2-C2-fmoc-20K]-[rIL-2] and 3-mer [mPEG2-CAC-fmoc-20K]-[rIL-2]were measured in a total IL-2 assay. Since the[mPEG2-C2-fmoc-20K]-[rIL-2] and [mPEG2-CAC-fmoc-20K]-[rIL-2] conjugatesare releasable conjugates, different species of the molecule will bepresent in a sample making individual quantitation difficult; therefore,total IL-2 levels were measured. The polymer-containing component of theconjugates was forced released from the conjugates by diluting thesamples and standard stock, prepared in neat plasma, 1:1 with releasingbuffer (100 mM HEPES/100 mM Tris-HCL, pH 9) and incubating at 37° C. for30 to 36 hours. After the incubations, a 25% volume of 0.1M acetic acidwas added to neutralize the high pH. The released IL-2 was measured bythe ELISA described above.

FIG. 9 shows a concentration-time plot of the tested articles in C57BL/6mice after a single, intra-muscular injection (1 mg/kg). Sodiumheparanized plasma samples were collected at ten minutes, and at 1, 6,24, 48, 72, 96, 120, 168 and 336 hours. The geomean concentrations werecalculated from 3 mice per time point. As shown in FIG. 9, aldesleukinhad a short ½-life and could not be detected after 6 hours (<0.05 ng/mL)while the conjugates had an extended ½-life and were still detected at336 hours.

Example 13 Lung Metastatic Melanoma Efficacy Studies

To evaluate the efficacy of compounds having purported IL-2 activity,the metastatic melanoma lung model has been widely used and is developedin C57BL/6 mice. In this model, mice are first intravenouslyadministered B16F10 melanoma cells, which causes the development of lungnodules of different numbers and sizes. The lung nodule numbers as wellas total surface area of these lesions varies depending on cellconcentrations implanted. A test compound of interest is thenadministered to a treatment group of mice and another group of mice isleft untreated to serve as a control. The efficacy of the test compoundcan be determined, as a percent reduction in the number and size of thelung nodules and the total lesion area for each lung between the treatedand untreated groups.

In this study, 100,000 B16F10 cells (passage not exceeding P8) wereimplanted by tail vein injections. On the third day from the date ofcell implantation, test compounds of interest (or vehicle) wereadministered as indicated in the Table 5 following either IP(intraperitoneal) or IV (intravenous) routes of administration.

TABLE 5 Groups Assignments for Example 13 Route Group B16F10 of admin-Animal no. Test Article cells istration No. Dose A aldesleukin 100,000IP 1-12 b.i.d × 5 (Prometheus Laboratories Inc.) B IL-2 moiety of100,000 IP 1-12 b.i.d × 5 Example 1 C vehicle 100,000 IP 1-12 b.i.d × 5D NKT-11135-A-001 100,000 IV 1-12 q2d × 3 E Pooled 3-mer/4-mer 100,000IV 1-12 q2d × 3 [mPEG2-CAC- fmoc-20K]- [rIL-2] F Pooled 1-mer/2-mer100,000 IV 1-12 q2d × 3 [mPEG2-ru-20K]- [rIL-2] G Pooled 3-mer/4-mer100,000 IV 1-12 q2d × 3 [mPEG2-ru-20K]- [rIL-2] Note: “b.i.d × 5” meanstwice a day for five days; “q2d × 3” means every second day for 3 doses

On day 14 from day of cell implantation, mice were sacrificed whileisolating and fixing lungs in the Bowen's solution containingformaldehyde for a day or two. The lungs (which were fixed in theBowen's solution) were examined under stereomicroscope and the numberand size of lesions for each lung were determined.

As shown in FIG. 10, on day 14 from the day of cell implantation micewere sacrificed and their isolated lungs were fixed in Bowen's solution.The tumor nodules and their sizes were counted for each of aldesleukin(Prometheus Laboratories Inc., San Diego Calif.), IL-2 moiety of Example1, pooled 3-mer/4-mer [mPEG2-CAC-fmoc-20K]-[rIL-2], pooled 3-mer/4-mer[mPEG2-ru-20K]-[rIL-2], and pooled 1-mer/2-mer [mPEG2-ru-20K]-[rIL-2].

Example 14 Subcutaneous B16F10 Melanoma Efficacy Studies

To evaluate the efficacy of compounds having purported IL-2 activity,the highly robust subcutaneous melanoma model in syngenic mice, i.e.,C57BL/6 mice, has been used. Briefly, one million B16F10 cells wereimplanted subcutaneously for each 5-6 week old C₅₇BL/6 mouse atmid-dorsal region. Tumors were allowed to grow to palpable size, i.e.,70-120 cu mm before randomization and assigning groups as shown in theTable 6. The mice were administered test compounds i.e., aldesleukin(Prometheus Laboratories Inc., San Diego Calif.), rIL-2-polymerconjugates or vehicle at different dose concentrations and dose regimes.The body weights and tumor volumes were measured every alternative day.The end point for this study is the time to reach median tumor volume of1500 cu mm for a given group or 45 days whichever is earlier.

TABLE 6 Groups Assignments for Example 14 Dose concen- Route Subgrouptration of admin- Test Compound (n = 9) (mg/kg) istration Dose Pooled3-mer/4-mer A1 2 IV q1d [mPEG2-CAC-fmoc-20K]- A2 4 [rIL-2] Pooled1-mer/2mer E1 6 IV q1d [mPEG2-C2-fmoc-20K]- E2 8 [rIL-2] aldesleukin(Prometheus C 3 IP b.i.d × 5 Laboratories Inc.) H (n = 6) Pooled3-mer/4-mer B1 2 IV q1d [mPEG2-C2-fmoc-20K]- B2 4 [rIL-2] Pooled1-mer/2-mer F1 6 IV q1d [mPEG2-ru-20K]- F2 9 [rIL-2] Pooled 1-mer/2-merG1 2 IV q1d [mPEG2-CAC-fmoc-20K]- G2 4 [rIL-2] Vehicle: 10 mM Sodium DAs per IV q1d acetate; 150 mM NaCl, I body pH 4.5; 2% Sucrose weight

Dose-response curves for tumor growth inhibition following theadministration of aldesleukin (Prometheus Laboratories Inc.) andrIL-2-polymer conjugates at different administration schemes areprovided in FIG. 11A and FIG. 11B. These results indicate that thetested rIL-2-polymer conjugates evidenced better efficacy at a singledose over aldesleukin (Prometheus Laboratories Inc.), which was dosed at3 mg/kg twice a day for five days.

FIG. 11A shows time to tumor progression after a single doseadministration of rIL-2-polymer conjugates to reach a median tumorvolume of 1500 mm³. The tumor growth delay (TGD) from the tumorprogression curves was found to be 4.6 and 6.2 days, respectively, forpooled 3-mer/4-mer [mPEG2-CAC-fmoc-20K]-[rIL-2] at 2 mg/kg and 4 mg/kgdose concentrations. For pooled 3-mer/4-mer [mPEG2-C2-fmoc-20K]-[rIL-2],TGD was found to be 6.4 and 7.6 days, respectively, at 2 mg/kg and 4mg/kg dose concentrations.

FIG. 11B shows time to tumor progression after single doseadministration rIL-2-polymer conjugates to reach a median tumor volumeof 1500 mm³. The TGD from the tumor progression curves was found to be3.6 and 4.6 days, respectively, for pooled 1-mer/2-mer[mPEG2-C2-fmoc-20K]-[rIL-2] at 6 mg/kg and 8 mg/kg dose concentrations.For pooled 1-mer/2-mer [mPEG2-ru-20K]-[rIL-2], the TGD was found to be3.8 at 2 mg/kg while a 4 mg/kg dose concentration was found to be toxicin nature. TGD from the tumor progression curves was found to be 2.2 and3.6 days, respectively, for pooled 1-mer/2-mer[mPEG2-CAC-fmoc-20K]-[rIL-2] at 2 mg/kg and 4 mg/kg dose concentrations.

In short, the efficacy in both a lung lesion metastasis model (Example13) and in a subcutaneous mouse melanoma model (Example 14) was achievedwith rIL-2 polymer conjugates at substantially lower frequency of dosingand lower overall protein amount as compared to aldesleukin (PrometheusLaboratories Inc.).

SEQ ID NO: 1 MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN-20        -10        1          11         21YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL31         41         51         61         71RPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS81         91         101        111        121 TLT SEQ ID NO: 2                      APTSSSTKKT QLQLEHLLLD LQMILNGINNYKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHLRPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT SEQ ID NO: 3                       PTSSSTKKT QLQLEHLLLD LQMILNGINNYKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHLRPRDLISNIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFSQSIIS TLT SEQ ID NO: 4                      APTSSSTKKT QLQLEHLLLD LQMILNGINNYKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHLRPRDLISRIN VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIIS TLT SEQ ID NO: 5  1 CATATGCCGACCAGCAGCAGCACCAAAAAAACCCAGCTGCAGCTGGAACATCTGCTGCTG  61GATCTGCAGATGATCCTGAACGGTATCAACAACTACAAAAACCCGAAACTGACCCGTATG 121CTGACCTTCAAATTCTACATGCCGAAAAAAGCAACCGAACTGAAACATCTGCAGTGCCTG 181GAAGAAGAACTGAAACCGCTGGAAGAAGTGCTGAACCTGGCACAGAGCAAAAACTTCCAT 241CTGCGTCCGCGTGATCTGATCAGCAACATCAACGTGATCGTGCTGGAACTGAAAGGTAGC 301GAAACCACCTTCATGTGCGAATACGCAGATGAAACCGCAACCATCGTGGAATTTCTGAAC 361CGTTGGATCACCTTCAGCCAGAGCATCATCAGCACCCTGACCTAAGAATTC

What is claimed is:
 1. A method comprising administering to anindividual a conjugate of an interleukin-2 moiety, the conjugatecomprising from one to seven branched poly(ethylene glycol) polymerseach covalently attached via a releasable linkage to an amino group ofan interleukin-2 moiety having the amino acid sequence of SEQ ID NO:1,wherein each branched poly(ethylene glycol) molecule has a weightaverage molecular weight from about 20,000 daltons to about 85,000daltons.
 2. The method of claim 1, wherein each branched poly(ethyleneglycol) polymer is terminally capped with C1-C5 alkoxy end-cappinggroups.
 3. The method of claim 2, wherein the alkoxy end-capping groupsare methoxy groups.
 4. The method of claim 1, wherein each branchedpoly(ethylene glycol) polymer comprises two poly(ethylene glycol)chains.
 5. The method of claim 1, wherein the releasable linkage is areleasable carbamate linkage.
 6. The method of claim 1, wherein eachbranched poly(ethylene glycol) polymer has a weight average molecularweight of about 20,000 daltons.
 7. The method of claim 1, wherein uponrelease of the branched poly(ethylene glycol) polymer in vivo, thepolymer detaches from the interleukin-2 moiety without leaving afragment thereof attached to the interleukin-2 moiety.
 8. The method ofclaim 1, wherein the conjugate has one, two, three or four branchedpoly(ethylene glycol) polymers each covalently attached via a releasablelinkage to an amino group of the interleukin-2 moiety.
 9. The method ofclaim 8, wherein the conjugate has one, two, or three branchedpoly(ethylene glycol) polymers each covalently attached via a releasablelinkage to an amino group of the interleukin-2 moiety.
 10. The method ofclaim 1, wherein the conjugate has four, five, six, or seven branchedpoly(ethylene glycol) polymers each covalently attached via a releasablelinkage to an amino group of the interleukin-2 moiety.
 11. The method ofclaim 10, wherein the conjugate possesses no interleukin-2 activityprior to release of the branched poly(ethylene glycol) polymer(s) fromthe conjugate in vivo following the administering.
 12. The method ofclaim 1, wherein the conjugate is comprised in a composition comprisinga plurality of the conjugates.
 13. The method of claim 12, wherein theplurality of conjugates is a mixture of conjugates selected from thegroup consisting of conjugates having four, five, six and seven of thebranched poly(ethylene glycol) polymers each covalently attached via areleasable linkage to an amino group of the interleukin-2 moiety. 14.The method of claim 12, wherein the plurality of conjugates is a mixtureof conjugates having an average of six branched poly(ethylene glycol)polymers per interleukin-2 moiety.
 15. The method of claim 1, whereinthe conjugate is comprised in a composition suitable for injection. 16.The method of claim 15, wherein the conjugate is administeredparenterally.
 17. The method of claim 16, wherein the conjugate isadministered intravenously.
 18. The method of claim 17, wherein theconjugate is administered to an individual having cancer.
 19. The methodof claim 18, wherein the cancer is selected from renal cell carcinoma,metastatic melanoma, acute myeloid leukemia, non-Hodgkin's lymphoma,cutaneous T-cell lymphoma, breast cancer and bladder cancer.
 20. Themethod of claim 1, wherein the conjugate is administered in combinationwith another active agent.